In this study, we isolated and tested restriction fragment lengthpolymorphism (RFLP) markers for Aspergillus fumigatus based on PCR products amplified by the random amplified polymorphic DNA (RAPD) primer R108. Four DNA fragments, Afd, Af5, Af4, and Af4A, were amplified. Fragments Afd and Af5 were 85% and 88% identical at the DNA level to part of the Afut1 retrotransposon from A. fumigatus. Fragment Af4A is a duplication of fragment Af4 and both showed similarity at the amino acid level with endonucleases from other fungal retrotransposons. We used both RAPD with primer R108 and RFLP assays with Afut1, Afd, and Af4A, to determine the genetic relatedness of clinical isolates of A. fumigatus isolated sequentially from four patients colonized with A. fumigatus. The combination of these different methods suggested that the isolates infecting the four patients were not identical.

Terminal restriction fragment lengthpolymorphism (T-RFLP) is an increasingly widely used technique in mycorrhizal ecology. In this paper, we review the technique as it is used to identify species of mycorrhizal fungi and distinguish two different versions of the technique: peak-profile T-RFLP (the original version) and database T-RFLP. We define database T-RFLP as the use of T-RFLP to identify individual species within samples by comparison of unknown data with a database of known T-RFLP patterns. This application of T-RFLP avoids some of the pitfalls of peak-profile T-RFLP and allows T-RFLP to be applied to polyphyletic functional groups such as ectomycorrhizal fungi. The identification of species using database T-RFLP is subject to several sources of potential error, including (1) random erroneous matches of peaks to species, (2) shared T-RFLP profiles across species, and (3) multiple T-RFLP profiles within a species. A mathematical approximation of the risk of the first type of error as a function of experimental parameters is discussed. Although potentially less accurate than some other methods such as clone libraries, the high throughput of database T-RFLP permits much greater replication and may, therefore, be preferable for many ecological questions, particularly when combined with other techniques such as cloning.

The restriction fragment lengthpolymorphism (RFLP) of the two human HLA-B-associated transcripts (BATs) genes, BAT1 and BAT2, was investigated using 5 different restriction enzymes and two human BAT1 and BAT2 cDNA probes. Two of the enzymes, NcoI and RsaI, revealed polymorphic patterns which were...

Full Text Available Background & objectives: The difficulties in diagnosis of neonatal sepsis are due to varied clinical presentation, low sensitivity of blood culture which is considered the gold standard and empirical antibiotic usage affecting the outcome of results. Though polymerase chain reaction (PCR based detection of bacterial 16S rRNA gene has been reported earlier, this does not provide identification of the causative agent. In this study, we used restriction fragment lengthpolymorphism (RFLP of amplified 16S rRNA gene to identify the organisms involved in neonatal sepsis and compared the findings with blood culture. Methods: Blood samples from 97 neonates were evaluated for diagnosis of neonatal sepsis using BacT/Alert (automated blood culture and PCR-RFLP. Results: Bacterial DNA was detected by 16S rRNA gene PCR in 55 cases, while BacT/Alert culture was positive in 34 cases. Staphylococcus aureus was the most common organism detected with both methods. Klebsiella spp. was isolated from four samples by culture but was detected by PCR-RFLP in five cases while Acinetobacter spp. was isolated from one case but detected in eight cases by PCR-RFLP. The sensitivity of PCR was found to be 82.3 per cent with a negative predictive value of 85.7 per cent. Eighty of the 97 neonates had prior exposure to antibiotics. Interpretation & conclusions:The results of our study demonstrate that PCR-RFLP having a rapid turnaround time may be useful for the early diagnosis of culture negative neonatal sepsis.

Terminal restriction fragment lengthpolymorphism (T-RFLP) analysis of PCR-amplified genes is a widely used fingerprinting technique in composting systems. This analysis is based on the restriction endonuclease digestion of fluorescently end-labeled PCR products. The digested product is mixed with a DNA size standard, itself labeled with a distinct fluorescent dye, and the fragments are then separated by capillary or gel electrophoresis using an automated sequencer. Upon analysis, only the terminal end-labeled restriction fragments are detected. An electropherogram is produced, which shows a profile of compost microbial community as a series of peaks of varying height. This technique has also been effectively used in the exploration of complex microbial environments and in the study of bacterial, archaeal, and eukaryal populations in natural habitats.

of using TGR gene variants as markers to distinguish among T. gondii isolates from different animals and different geographical sources. Based on the band patterns obtained by restriction fragment lengthpolymorphism (RFLP) analysis of the polymerase chain reaction (PCR) amplified TGR sequences, the T...

Oligonucleotide primers were designed from the conserved nucleotide sequences of 18S ribosomal RNA (18S rRNA) gene of protozoans: Trypanosoma brucei, Leishmania donovani, Triponema aequale and Lagenidium gigantum. The primers were used in polymerace chain reaction (PCR) to generate PCR products of approximately 1 Kb using genomic DNA from T. brucei and the four genotypic groups of T. congolense as template. The five PCR products so produced were digested with several restriction enzymes and hybridized to a DNA probe made from T. brucei PCR product of the same 18S rRNA gene region. Most restriction enzyme digests revealed polymorphism with respect to the location of their recognition sites on the five PCR products. The restriction fragment lengthpolymorphism (RFLP) pattern observed indicate that the 18S rRNA gene sequences of trypanosomes: T. brucei and the four genotypes of T.congolence group are heterogeneous. The results further demonstrate that the region that was amplified can be used in specific identification of trypanosomes species and subspecies.(author)

Full Text Available Restriction fragment lengthpolymorphism (RFLP analysis was developed for genetic typing of Polish strains of bovine viral diarrhoea virus (BVDV. The method was applied using 60 BVDV isolates, which included BVDV genotype 1, subtypes a, b, d, e, f, and g, and genotype 2a. RT-PCR products of the 5’untranslated region (5’UTR were digested using three enzymes. Restriction patterns classified the strains into seven groups, each with a specific and different pattern from other subtypes. These findings were confirmed by nucleotide sequencing and phylogenetic analysis. The results suggest that RFLP analysis is a simple, reliable, and fast genotyping method for BVDV strains in comparison with sequencing. This method can distinguish six subtypes of BVDV-1 including a new subtype 1e, identified exclusively by this method, and it allows differentiation of BVDV-1 from BVDV-2 genotype.

We have used a recombinant DNA library constructed from flow-sorted human chromosome 8 as a source of single-copy human probes. These probes have been screened for restriction fragment lengthpolymorphism (RFLP) by hybridization to Southern transfers of genomic DNA from five unrelated individuals. We have detected six RFLPs distributed among four probes after screening 741 base pairs for restriction site variation. These RFLPs all behave as codominant Mendelian alleles. Two of the probes detect rare variants, while the other two detect RFLPs with PIC values of .36 and .16. Informative probes will be useful for the construction of a linkage map for chromosome 8 and for the localization of mutant alleles to this chromosome. Images Fig. 1 PMID:2879441

Full Text Available The milk is an important food because it contents Conjugated Linoleic Acids (CLA. These fatty acids are synthesized in mammary gland under action of the enzyme Stearoyl CoA-Desaturase (SCD and have showed some positive effects in human disease prevention and treatments. A variation of CLA in milk fat exists and can be partially explained by the different levels of expression of SCD. The aim was to study part of the encoding regions of SCD´s gene using PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment LengthPolymorphism. Genomic DNA was extracted from lactating Murrah females. After this, PCR reactions were made by using primers Z43D1 that encloses exon I, II and intron I. The fragments amplified are composed by 938 pb. Then, RFLP techniques were applied in the fragments using the restriction enzymes Pst I and Sma I. The enzyme Pst I has generated fragments of 788pb and 150bp and the Sma I has generated fragments of 693pb and 245pb. All the animals showed the same migration standard for both enzymes, characterizing a genetic monomorphism for this region of SCD gene. The analysis determined that there aren’t genetic differences between these animals in the studied regions by using Pst I and Sma I enzymes.

To establish a new polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP) method of genotyping HBV using Mbo I, BsTN I, BsmA I, Hpa II and investigate the relationship between genotype and clinical spectrum of hepatitis B. 124 full-genomic HBV sequences and 13 S-genomic sequences were analyzed, genotype specific regions were identified by the restriction enzymes Mbo I, BsTN I, BsmA I, Hpa II. And 176 samples from different kinds of hepatitis B were genotyped by this method. Five samples had been randomly selected and directly sequenced their S gene, to assess the accuracy. In 176 serum samples of patients with hepatitis B from Guizhou area, genotype B and C were found in 56.8% and 43.2% respectively. The proportions of genotype B and C in ASC were 40.0% and 15.7% (chi-square = 12.16, P < 0.005); and they were 31.6% and 14.0% in CHB (chi-square = 7.88, P < 0.005). Genotyping HBV, based on S gene RFLP seems to be highly sensitive, differential and accurate and could be used in large-scale surveys. HBV genotype B and C are existed in Guizhou area.

In this paper, we describe the identification of highly polymorphicRFLP loci and their application to genotyping in humans and to mapping the CF gene to chromosome 7. We also report the construction of a high-resolution genetic map of chromosome 7 and summarize progress toward the development of a presymptomatic diagnostic test for CF that should be useful in virtually every case. 25 references, 7 figures, 5 tables

The restriction fragment lengthpolymorphism of the human tumour necrosis factor (TNF alpha) region was investigated by means of 20 different restriction enzymes and a human TNF alpha cDNA probe. Only one of the enzymes, NcoI, revealed a polymorphic pattern consisting of fragments of 10.5 and 5...

The restriction fragment lengthpolymorphism of the human tumour necrosis factor (TNF alpha) region was investigated by means of 20 different restriction enzymes and a human TNF alpha cDNA probe. Only one of the enzymes, NcoI, revealed a polymorphic pattern consisting of fragments of 10.5 and 5.5...

A rapid method was developed for partial characterization of the replicase-encoding open reading frame 1 (ORF 1) of porcine reproductive and respiratory syndrome virus (PRRSV). It comprised long RT-PCR amplification of 11.1 kb (94%) of ORF 1, followed by restriction fragment lengthpolymorphism a...

Comparison of usefulness of IS1245 RFLP and VNTR in M. avium genotyping. Thirty-six cases (55 strains) from sputum and BALF and twelve cases (29 strains) isolated from blood of HIV-infected patients were used. VNTR and RFLP using IS1245 were performed. Multiple samples were taken from 16 patients and 52 clinical isolates were used for VNTR and RFLP for comparison. (1) VNTR and RFLP results were identical in 12 out of 16 cases whose samples were collected several times. (2) Eight isolates were obtained from one patient. In this eight isolates, there were the cases of M. avium polyclonal infection and of mixed infection with M. intracellulare. VNTR patterns were two types and RFLP were 5 kinds of different in this case. (3) VNTR patterns of six isolates from one HIV-infected patient were identical, but there were three variations in RFLP patterns. There were three cases of mixed infections with M. tuberculosis or M. intracellulare, and six strains polyclonal infection of M. avium (7.1 %) in 84 isolates. These 6 clinical isolates were derived from sputum or BALF (5 strains) and HIV-infected blood (one strain). VNTR patterns were similar in four pairs (9 strains) who did not contact closely, but they were distinguished clearly by RFLP. Seventeen strains had three or less IS1245-related bands in RFLP analyses of 89 strains. As there is a possibility of polyclonal infection with M. avium and mixed infection with other species, the single clonal infection should be confirmed first by VNTR. When single colony was obtained, VNTR and RFLP were performed for genotyping of M. avium. Furthermore, strains with less bands by RFLP should be carefully judged in terms of both VNTR and RFLP. It is recommended that the specimens should be collected from each patient several times.

To develop a proper assay for identifying single nucleotide polymorphisms( SNPs) of the MGMT gene. PCR primers were designed by create restriction site (CRS) method, then polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP) was adopted to identify four SNPs in MGMT gene. By PCR, one primer pair yielded target products containing MGMT84 SNP site, and the other primer pair yielded target products containing MGMT143, 160, 178 SNP sites. Four restriction enzymes were adopted to identify the four SNPs, respectively. The effects of PCR and RFLP were good. The methods for four SNPs of MGMT determinated by CRS-PCR-RFLP theory could be facility, economy, and rapidness.

A rapid method was developed for partial characterization of the replicase-encoding open reading frame 1 (ORF 1) of porcine reproductive and respiratory syndrome virus (PRRSV). It comprised long RT-PCR amplification of 11.1 kb (94%) of ORF 1, followed by restriction fragment lengthpolymorphism...... analysis. The method was used to compare ORF 1 sequences of two divergent European-type PRRSV strains. Our results indicated that the structural and replicase parts of these two strains had evolved at overall similar rates....

The X-linked immunodeficiency (Xid) in CBA/N mice serves as a model for the X-linked agammaglobulinemia (XLA) syndrome in man. X-chromosome inactivation in F{sub 1} heterozygotes derived from CBA/N (X{sup xid}/X{sup xid}) and B6.Pgk-1a (X{sup +}/Y) was investigated by monitoring the methylation status of the individual Pgk-1 alleles, Pgk-1b and Pgk-1a, respectively, using a novel Tth111I RFLP. Results indicate that in circulating B lymphocytes of female heterozygotes, only the X chromosomes carrying the normal alleles (X{sup +}) are active (nonrandom inactivation of the X chromosome), whereas in non-B cells both the X chromosomes (X{sup +} and X{sup xid}) are active (random inactivation of the X chromosome). These results were further confirmed by direct evaluation of transcription of the Btk gene, the gene mutated both in Xid and in XLA. 36 refs., 2 figs., 2 tabs.

Intracellular folate homeostasis and metabolism is regulated by numerous genes. Among them, 5,10-methylenetetrahydrofolate reductase (MTHFR) is of special interest because of its involvement in regulation of the homocysteine level in the body as a result of folate metabolism. Moreover, some studies demonstrated that the homocysteine plasma level in individuals may be influenced by polymorphisms present in the MTHFR gene. Two common, clinically relevant mutations have been described: MTHFR C677T and MTHFR A1298C. Although several laboratory techniques allow genotyping of both polymorphisms, PCR-RFLP analysis is simple to perform, relatively cheap, and thus one of the most utilized. In the case of A1298C, the PCR-RFLP technique that utilizes MboII endonuclease class II requires an acrylamide gel electrophoresis, since agarose gel electrophoresis is unable to resolve short deoxyribonucleic acid (DNA) fragments after restriction digestion. Agarose gel electrophoresis is commonly preferred over that of acrylamide. To resolve this inconvenience, a novel PCR-RFLP, AjuI-based method to genotype A1298C alleles has been developed that can be performed on standard agarose gel.

We used the polymerase chain reaction-restriction fragment lengthpolymorphism (PCRRFLP) technique to screen for DNA polymorphism in 109 cattle. In all cattle, we amplified an 863 fragment consisting of part of exon 8. The amplified fragment digested with HaeIII restriction endonuclease and subjected to electrophoretic ...

The restriction fragment lengthpolymorphism (RFLP) pattern of PCR products obtained was the same for T. brucei subspecies: T.b. brucei and T.b. gambiense but different for other trypanosome species and L. donovani. RFLP analysis was also done with genomic DNA from different trypanosome species, subspecies and ...

Protection of telomeres protein 1 (POT1) plays pivotal roles in protection of chromosome ends and regulation of telomere length with other telomere binding proteins; its genetic polymorphisms are associated with many diseases. In this study, we explored a novel PCR-RFLP method for typing the single nucleotide polymorphism (SNP) rs1034794 of the human POT1 gene. A new restriction enzyme site was introduced into a POT1 gene amplification product by created restriction site PCR (CRS-PCR). One primer was designed based on changed sequence; after PCR amplification, a new restriction enzyme site for AluI was introduced into the PCR products. One hundred and seventy eight samples from Han Chinese individuals were tested to evaluate this new method. The 3'-end of the forward primer was next to the polymorphic site, and the third base from the 3'-end was the mismatched base A. The final PCR product contained the AGCT sequence (AluI recognition site) when the ancestral POT1 alleles were amplified. The data obtained with the new method perfectly matched those obtained with the sequencing method. Thus, CRS-PCR is a new low-cost and high-efficiency alternative for rs1034794 typing.

Growth dynamics of Pseudomonas aeruginosa, Burkholderia cepacia, and Staphylococcus aureus in a batch and chemostat, were investigated as a laboratory model system for persistent infections in cystic fibrosis. Most species-specific enumeration methods for mixed cultures are laborious or only qualitative, and therefore impede generation of quantitative data required for validation of mathematical models. Here, a quantitative T-RFLP method was evaluated and applied for specific and absolute cell number enumerations. The method was tested to be unbiased by quantitative sample composition and allowed reproducible enumerations of mixed cultures. For assay validation, samples of defined concentration containing one, two or three species were quantified. Logarithmically transformed absolute cell numbers of single-species dilutions were linear within a lower working range of 10(4)-10(6) cfu/mL (species-dependent) and an upper working range of 10(10) cfu/mL. Quantifications of single species (10(6)-10(10) cfu/mL) spiked with one or two other species agreed well with single species controls. Differences between slopes of first order linear regression of spiked and pure dilution series were insignificant. Coefficient of variation of defined mixed replicates was maximum 4.39%, of a three-species chemostat it was maximum 1.76%. T-RFLP monitoring of pure cultures in parallel shake flasks and of a three-species mixed chemostat gave very consistent results. Coexistence of at least two species after a time period equivalent to more than 33 volume exchanges was found. This result was not predicted from pure cultures clearly indicating the need for quantitative mixed culture experiments to better understand microbial growth dynamics and for mathematical model validation.

The allelic frequency of any polymorphism within a population determines its usefulness for genetic counselling. This is important in populations of non-Caucasian origin as RFLPs may significantly differ among ethnic groups. We report a study of five intragenic polymorphisms in factor VIII gene carried out in Singapore. The three PCR-based RFLP markers studied were Intron 18/Bcl I, Intron 19/Hind III and Intron 22/Xba I. In an analysis of 148 unrelated normal X chromosomes, the allele frequencies were found to be A1 = 0.18, A2 = 0.82 (Bcl I RFLP), A1 = 0.80, A2 = 0.20 (Hind III RFLP) and A1 = 0.58, and A2 = 0.42 (Xba I RFLP). The heterozygosity rates of 74 females analyzed separately were 31%, 32% and 84.2%, respectively. Linkage disequilibrium was also observed to some degree between Bcl I and Hind III polymorphism in our population. We have also analyzed a sequence polymorphism in Intron 7 using hybridization with radioactive-labelled {sup 32}P allele-specific oligonucleotide probes. This polymorphism was not very polymorphic in our population with only 2% of 117 individuals analyzed being informative. However, the use of a hypervariable dinucleotide repeat sequence (VNTR) in Intron 13 showed that 25 of our of 27 (93%) females were heterozygous. Allele frequencies ranged from 1 to 55 %. We conclude that a viable strategy for molecular analysis of Hemophilia A families in our population should include the use of Intron 18/Bcl I and Intron 22/Xba I RFLP markers and the Intron 13 VNTR marker.

Calpastatin has been known as candidate gene in muscle growth efficiency and meat quality. This gene has been located to chromosome 5 of sheep. In order to evaluate the calpastatin gene polymorphism, random blood sample were collected from 111 Arabic ram sheep from different regions. The DNA extraction was ...

are associated with failure of sulpha prophylaxis and increased mortality in HIV-1 positive patients with PCP, suggesting that DHPS mutations may cause sulpha resistance. To facilitate detection of DHPS mutations we developed a restriction fragment lengthpolymorphism (RFLP) assay, detecting mutations at codon...

Species identification is a basic issue in biosecurity. Polymerase chain reaction (PCR) followed by restriction fragment lengthpolymorphism (RFLP) is a useful molecular diagnostic tool for species identification. However, the lack of transferability of data has been a serious shortcoming of this method. A RFLP catalog, i.e., a graph of PCR-RFLP patterns expected from sequence data, was devised as a tool to facilitate PCR-RFLP data sharing among laboratories. Twelve species of Tetranychus spider mites have been recorded in Japan to date. In this study, we analyzed DNA sequences of the internal transcribed spacer (ITS) region in nuclear ribosomal DNA of 11 Tetranychus species. For the species identification using PCR-RFLP, we chose six candidates from 131 restriction endonucleases and developed an RFLP catalog of all known Japanese Tetranychus species except Tetranychus neocaledonicus André. The RFLP catalog revealed that most Tetranychus species had diagnostic restriction fragments. The RFLP catalog is transferable and simple molecular diagnostic tool, and it has the ability to add more species and newly found intraspecific variations. Therefore, we believe that the RFLP catalog will contribute to biosecurity as a practical diagnostic tool for species identification of spider mites.

To develop a appropriate assay for identifying single nucleotide polymorphism (SNP) of alcohol dehydrogenase 2 (ADH2) gene. According to base substitution situation of one single base mutational site, we designed the present study primers. One of the primers was designed on the basis of neighbourhood sequence of the mutational site, that is, we made one mismatch base to let product a new enzyme site between the 3' end of the primer and the single base mutation type after the PCR amplification. Then PCR-RFLP was adopted to identify the SNP in ADH2 gene. One primer pair can get target products containing ADH2 SNP site by PCR, restriction enzymes Bsh1236I were adopted to identify the SNP site. The expected results were reached. It suggested that the method of detecting the SNP of ADH2 based on CRS-PCR-RFLP theory is facilitated, economic, and rapid.

To determine the rate at which IS6110 restriction fragment lengthpolymorphism (RFLP) patterns in Mycobacterium tuberculosis change over time, we applied a smooth nonparametric survival model to several data sets, including data from previous publications on the rate of change. The results strongly

DNA restriction fragment lengthpolymorphisms (RFLPs) are used for diagnosis of genetic disease in families known to be affected by specific disorders, but RFLPs can be also useful for the differential diagnosis of hereditary disease. An RFLP pattern represents the inheritance of chromosomal markers

We have used the polymerase chain reaction (PCR) in combination with the restriction fragment lengthpolymorphism (RFLP) technique for HLA-DBP1 typing. After PCR amplification of the polymorphic second exon of the HLA-DPB1 locus, the PCR product was digested with seven allele-specific restriction...

Full Text Available AbstractThe aim of this study was detection of polymorphism in the bovine growth hormone gene using PCR – RFLP asan efficient method for genetic analysis of polymorphism. A polymorphic site of the growth hormone gene (AluIpolymorphism that results in an amino acid change at position 127 of the protein chain (leucine, L to valine, V hasbeen linked to differences in circulating metabolites and metabolic hormones and to milk yield traits. The growthhormone gene is a candidate gene for body weight and weight gain in cattle since it plays a fundamental role ingrowth regulation. The polymorphism of growth hormone gene was studied in a group of 58 bulls of Slovak spottedbreed. A strategy employing PCR was used to amplify 428 bp products from blood samples. Digestion of PCRproducts with restriction enzyme AluI revealed two alleles: allele L was 265, 96, 51, 16 fragments and allele V was265, 147 and 16. Three patterns were observed and with frequencies 0.404, 0.473 and 0.123 for LL, LV and VV,respectively. The frequency of alleles L was 0.6404 and V was 0.3596.

Fifteen Coccidioides isolates were previously examined for genetic diversity using restriction fragment lengthpolymorphism (RFLP); two fragment patterns were observed. Two isolates demonstrated one banding pattern (designated RFLP group I), while the remaining 13 isolates demonstrated a second pattern (designated RFLP group II). Recently, molecular studies supported the division of the genera Coccidioides into two species: Coccidioides posadasii and Coccidioides immitis. It has been assumed that the species division corresponds to the RFLP grouping. We tested this hypothesis by amplifying the ribosomal DNA internal transcribed spacer region as well as the dioxygenase, serine proteinase, and urease genes from 13 isolates previously examined by RFLP and then sequencing the PCR products. The appropriate species for each isolate was assigned using phylogenetically informative sites. The RFLP grouping agreed with the Coccidioides species assignment for all but one isolate, which may represent a hybrid. In addition, polymorphic sites among the four genes examined were in agreement for species assignment such that analysis of a single gene may be sufficient for species assignment.

Complementary DNA-amplified fragment lengthpolymorphism (AFLP-cDNA) analysis of differential gene expression from the xerophyte Ammopiptanthus mongolicus in response to cold, drought and cold together with drought.

Full Text Available Restriction fragment lengthpolymorphism (RFLP was used to examine the extent of mtDNA polymorphism among six strains of rats (Rattus norvegicus - Wistar, Wistar Munich, Brown Norway, Wistar Kyoto, SHR and SHR-SP. A survey of 26 restriction enzymes has revealed a low level of genetic divergence among strains. The sites of cleavage by EcoRI, NcoI and XmnI were shown to be polymorphic. The use of these three enzymes allows the 6 strains to be classified into 4 haplotypes and identifies specific markers for each one. The percentage of sequence divergence among all pairs of haplotypes ranged from 0.035 to 0.33%, which is the result of a severe population constriction undergone by the strains. These haplotypes are easily demonstrable and therefore RFLP analysis can be employed for genetic monitoring of rats within animal facilities or among different laboratories.

Objectives: In this study we have evaluated the mutagenicity of organophosphate pesticides acephate, chlorpyrifos, and profenofos using polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP) assay with the mosquito Culex quinquefasciatus taken as an experimental model. Materials and Methods: Second instar larvae were treated with LC20 of each pesticide for 24 h and mutations induced in the sequence of mitochondrial COII gene (690bp) were studied from restriction pattern...

A high-performance multi-analysis system for genotypic mutation by means of restriction fragment lengthpolymorphisms (RFLP) involving endonuclease treatment of PCR-amplified DNA on a microchip and subsequent analysis by microchip electrophoresis for DNA sizing was developed. A Hitachi SV1210 system, with which 12 samples can be analyzed on a plastic chip with good accuracy as to DNA sizing between 25 and 300 bp, was employed for RFLP analysis. We performed RFLP analysis of the ABO genotypes of blood donors for whom the ABO type was known. Six blood samples were analyzed by PCR to amplify two different regions of the genomic DNA, each of the amplified DNAs containing a different nucleotide polymorphism. To analyze the genes at polymorphic sites 261 and 526, restriction endonucleases Kpn I and Ban I were employed, respectively. When an amplified DNA was digested with each endonuclease on a microchip for 20 min, sequential analysis revealed the presence or absence of the respective restriction site. This analysis was performed within 7 min using a 1/10 volume of a DNA sample in comparison with the conventional method, and the estimated DNA size differed from the predicted size by less than 10 bp. The results indicate the potential of microchip electrophoresis for RFLP with on-chip direct endonuclease digestion and sequential analysis, offering high resolution in a short time.

May 9, 2011 ... Complementary DNA-amplified fragment lengthpolymorphism (cDNA-AFLP) technology was used to analyze ... that 9 of the studied expressed sequence tags (ESTs) are related to protein modification, 12 ESTs are involved in the .... primers were used during the first strand synthesis of our cDNA synthesis ...

Traditional methods of identifying microorganisms through culturing and microscopy techniques can be somewhat tedious and time consuming. A faster and more accurate method for identifying microorganisms is through the sequencing of its ribosomal gene. Classification of microorganisms by ribosomal gene sequencing has become widely accepted within the scientific community. Although this method is quite definitive in its ability to identify the microorganism being studied, it usually involves a pure culture and then the cloning and sequencing of its ribosomal gene. In order to look at complex communities and uncultured microorganisms, many researches have removed the culturing step and moved towards the generation of 16S clone libraries. Data generated from numerous 16S clone libraries from countless environments have produced databases full of ribosomal sequences that may have never been gathered if culturing of the microorganism had been a prerequisite. Ribosomal clone libraries are still quite time consuming, especially if one is interested in detecting differences between complex community structures under varying conditions, such as the effect, diet can impose on the rumen microbial community. Rapid screening methods that allow for the presentation of phylogenetic ribosomal diversity patterns from complex communities in an easy-to-interpret and reproducible manner have all benefited from the knowledge gained from ribosomal clone libraries. Restriction fragment lengthpolymorphism (RFLP) and terminal restriction fragment lengthpolymorphism (T-RFLP) are two such techniques that will be described in this chapter. Other methods such as ribosomal intergenic spacer analysis (RISA), which determines diversity through differences, found in the transcribed spacer region between the highly conserved ribosomal genes can also be employed. RISA is a particularly powerful tool for attempting to discriminate between closely related species and strains. Two methods that do not

Molecular identification of puerulus larvae of Panulirus homarus of the genus Panulirus from Indian coast was studied by employing Polymerase Chain Reaction, Restriction Fragment LengthPolymorphism (PCR-RFLP) analysis of the mitochondrial DNA (mtDNA) Cytochrome Oxidase Gene (COI) by agarose gel electrophoresis and Denaturing Gradient Gel Electrophoresis (DGGE). The size of amplified fragment of COI gene was estimated to be approximately 1300 base pairs (bp). Single fragment amplification was recorded during different stages of the life cycle. The RFLP digestion was carried out using five different restriction enzymes (BsplI, HhaI, RsaI, TaqI and AluI). The RFLP profile of the different endonucleases, varied between 1-5 restriction types. RFLP analysis using endonuclease TaqI enabled identification of P. homarus during different stages of its life history.

Different molecular methods for the discrimination of Candida glabrata, C. bracarensis and C. nivariensis were evaluated and the prevalence of these species among Danish blood isolates investigated. Control strains were used to determine fragment lengthpolymorphism in the ITS1, ITS2, ITS1-5.8S...... enzymes were suitable for RFLP differentiation of the species. Enzymatic digestion of the D1/D2 domain with TatI produced unique band sizes for each of the three species. PCR-RFLP and PNA-FISH were in agreement for all of the isolates tested. None of the 133 Danish blood isolates were C. nivariensis or C....... bracarensis. Fragment size polymorphism of ITS1 and RFLP of the D1/D2 domain or the ITS region are useful methods for the differentiation of the species within the C. glabrata group. C. bracarensis and C. nivariensis are rare among Danish C. glabrata blood isolates....

The aim of study was to identify the association of FSH beta-subunit gene polymorphisms with sperm quality traits. A total of 470 samples of normal mature bull from several breeds were used for population study and 127 bulls from National and Regional AI centre of Indonesia for association study. To amplify, a PCR-RFLP method was used and digested with Pst1 restriction enzyme. The allele frequency of the A and B in Bali cattle were (0.000) and (1.000), respectively. The absence of other allel...

In 2009, 3 genome-wide association studies implicated IL28B single-nucleotide polymorphisms (SNPs) as the strongest genetic pretreatment predictor of sustained virological response (SVR) in hepatitis C infection. Recently, the American Association for the Study of Liver Diseases (AASLD) and the European Association for the Study of the Liver (EASL) included IL28B testing in their guidelines. The main aim of this study was to develop and validate a simple, rapid, and inexpensive polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP) method for genotyping of common IL28B polymorphisms (rs12979860 and rs8099917). Two methods were developed to genotype common IL28B polymorphisms: 1) PCR-sequencing as a reference method and 2) PCR-RFLP as a rapid and inexpensive method. Both polymorphisms were genotyped in 104 Iranian hepatitis C patients by both methods simultaneously. To validate the PCR-RFLP method, the PCR-RFLP genotyping results should be 100% concordant with the PCR-sequencing results. Genotyping of rs12979860 and rs8099917 by PCR-RFLP was concordant with PCR-sequencing in 104 (100%) individuals. The analytical sensitivity and specificity of the PCR-RFLP method for genotyping of both SNPs are 100%. Among these 104 patients with chronic hepatitis C, the frequency of the rs12979860 CC, CT and TT genotypes were 40.4%, 47.1% and 12.5% and the frequency of the rs8099917 TT, GT and GG genotypes were 59.6%, 35.6% and 4.8%, respectively. Also, three IL28B haplotypes (rs12979860-rs8099917) were found among our patients including C-T, T-G and T-T with 63.9%, 22.6% and 13.5% frequency, respectively. C-G haplotype was absent in all of our patients. We have developed a validated, fast, and simple PCR-RFLP method for genotyping of common IL28B SNPs that is more cost-effective than sequencing.

Full Text Available The aim of study was to identify the association of FSH beta-subunit gene polymorphisms withsperm quality traits. A total of 470 samples of normal mature bull from several breeds were used forpopulation study and 127 bulls from National and Regional AI centre of Indonesia for association study.To amplify, a PCR-RFLP method was used and digested with Pst1 restriction enzyme. The allelefrequency of the A and B in Bali cattle were (0.000 and (1.000, respectively. The absence of otherallele A suggested that the Bali cattle was monomorphic, while Brahman, FH, Simmental and Limousinewere polymorphic. The highest observed heterozygosity were found in Limousine (0.318 and thehighest expected heterozygosity were in Simmental (0.420. The higher incident of percentage of spermabnormalities were found in Simmental, Limousin, Brahman compared to Bali and FH. Among all typesof sperm abnormalities, the abaxial and microcephalus were found in highest number.

Various molecular techniques like analysis of the amplified rDNA internal transcribed spacers (ITS), intragenic spacers and total ITS region analysis by restriction fragment lengthpolymorphism (RFLP) has been introduced for yeast identification but there are limited databases to identify yeast species on the basis of 5.8S rDNA. In this study, twenty nine yeast strains from various sources including spoiled fruits, vegetables, foodstuffs, and concentrated juices were characterized by PCR-RFLP. PCR-RFLP has been used to characterize yeasts present in different spoiled food samples after isolation of the yeasts. By using this technique, the isolated yeast strains were characterized by direct 5.8S-ITS rDNA region amplification. RFLP analysis was applied to each of the amplification products (varied from 400bp to 800bp) detected, and the corresponding yeast identifications were made according to each specific restriction patterns obtained after treatment with two endonucleases TaqI and HaeIII which yielded a specific banding pattern for each species. For further confirmation amplified products of eleven selected isolates were sequenced and blast on NCBI. Both RFLP and sequence analyses of the strains with accession nos. KF472163, KF472164, KF472165, KF472166, KF472167, KF472168, KF472169, KF472170, KF472171, KF472172, KF472173 gave significantly similar results. The isolates were found to belong five different yeast species including; Candida spp., Pichia spp., Kluyveromyces spp., Clavispora spp. and Hanseniaspora spp. This method provides a fast, easy, reliable and authentic way for determining yeast population present in different type of samples, as compared to traditional characterization technique. (author)

New chicken Rfp-Y haplotypes were determined by the use of restriction fragment lengthpolymorphism (RFLP) and mixed lymphocyte culture (MLC) in four different chicken haplotypes, B15, B19, B21, B201. The RFLPpolymorphism was mapped to the Rfp-Y system by the use of a subclone (18.1) which maps...

Full Text Available Amplified Fragment LengthPolymorphism molecular markers (AFLPs has been developed combining procedures of RFLPs and RAPDs molekular markers, i.e. the first step is restriction digestion of the genomic DNA that is followed by selective amplification of the restricted fragments. The advantage of the AFLP technique is that it allows rapid generation of a large number of reproducible markers. The reproducibility of AFLPs markers is assured by the use of restriction site-specific adapters and adapter-specific primers for PCR reaction. Only fragments containing the restriction site sequence plus the additional nucleotides will be amplified and the more selected nucleotides added on the primer sequence the fewer the number of fragments amplified by PCR. The amplified products are normally separated on a sequencing gel and visualized after exposure to X-ray film or by using fluorescent labeled primers. AFLP shave proven to be extremely proficient in revealing diversity at below the species level. A disadvantage of AFLP technique is that AFLPs are essentially a dominant marker system and not able to identify heterozygotes.

In comparison to restriction fragment lengthpolymorphism (RFLP) typing, variable number of tandem repeat (VNTR) typing is easier to perform, faster and yields results in a simple, numerical format. Therefore, this technique has gained recognition as the new international gold standard in typing of

BACKGROUND: In comparison to restriction fragment lengthpolymorphism (RFLP) typing, variable number of tandem repeat (VNTR) typing is easier to perform, faster and yields results in a simple, numerical format. Therefore, this technique has gained recognition as the new international gold standard

Full Text Available Abstract Background In comparison to restriction fragment lengthpolymorphism (RFLP typing, variable number of tandem repeat (VNTR typing is easier to perform, faster and yields results in a simple, numerical format. Therefore, this technique has gained recognition as the new international gold standard in typing of Mycobacterium tuberculosis. However, some reports indicated that VNTR typing may be less suitable for Beijing genotype isolates. We therefore compared the performance of internationally standardized RFLP and 24 loci VNTR typing to discriminate among 100 Beijing genotype isolates from the Southern Vietnam. Methods Hundred Beijing genotype strains defined by spoligotyping were randomly selected and typed by RFLP and VNTR typing. The discriminatory power of VNTR and RFLP typing was compared using the Bionumerics software. Results Among 95 Beijing strains available for analysis, 14 clusters were identified comprising 34 strains and 61 unique profiles in 24 loci VNTR typing ((Hunter Gaston Discrimination Index (HGDI = 0.994. 13 clusters containing 31 strains and 64 unique patterns in RFLP typing (HGDI = 0.994 were found. Nine RFLP clusters were subdivided by VNTR typing and 12 VNTR clusters were split by RFLP. Five isolates (5% revealing double alleles or no signal in two or more loci in VNTR typing could not be analyzed. Conclusions Overall, 24 loci VNTR typing and RFLP typing had similar high-level of discrimination among 95 Beijing strains from Southern Vietnam. However, loci VNTR 154, VNTR 2461 and VNTR 3171 had hardly added any value to the level of discrimination.

Conventional identification of mycobacteria is achieved by standard biochemical tests that are time consuming, laborious and is not always conclusive. This study was thus undertaken to standardize a simple, rapid and cost-effective polymerase chain reaction based restriction fragment lengthpolymorphism (PCR-RFLP) using primers coding for the 16S - 23S rRNA spacer region to identify the mycobacterial isolates to the species level. The PCR with primers targeting the 16S-23S rRNA spacer region was standardized using the standard mycobacterial strains and applied on 51 clinical isolates. The PCR amplified products were subjected to RFLP using the restriction enzymes, Hae III, MspI and BstXI. The results obtained were compared with those of conventional biochemical tests. PCR was sensitive to detect 2.5 pg of H37Rv DNA (370 bp for slow grower mycobacteria) and 1.5 pg of M. fortuitum DNA (450 bp for rapid grower mycobacteria). Based on the PCRRFLP products obtained the 51 mycobacterial isolates were classified into 41 slow growers and 10 rapid growers. Among the 41 slow growers, 40 were identified as M. tuberculosis, one as M. xenopi and 10 rapid growers as M. fortuitum. PCR using primers targeting the 16S-23S rRNA spacer region was a reliable tool for rapid identification of mycobacterial isolates into slow and rapid growers within 4 h of isolation and further speciation by PCR-RFLP within 6-8 h.

The main aim of this study was to contribute to the mapping of the human genome by searching for and characterizing a number of RFLPs (restriction fragment lengthpolymorphisms) in the human genome. The more specific aims of this study were: 1. To isolate single-copy human DNA sequences from a human genomic library. 2. To use these single-copy sequences as DNA probes to search for polymorphic variation among Caucasoid individuals. 3. To show by means of family studies that the RFLPs were inherited in a co-dominant Mendelian fashion. 4. To determine the population frequencies of these RFLPs in Southern African Populations, namely the Bantu-speaking Negroids and the San. 5. To assign these RFLP-detecting DNA sequences to human chromosomes using somatic cell hybrid lines. In this study DNA was labelled with Phosphorus 32

In order to switch from IS6110 and polymorphic GC-rich repetitive sequence (PGRS) restriction fragment lengthpolymorphism (RFLP) to 24-locus variable-number tandem-repeat (VNTR) typing of Mycobacterium tuberculosis complex isolates in the national tuberculosis control program in The Netherlands, a

In order to switch from IS6110 and polymorphic GC-rich repetitive sequence (PGRS) restriction fragment lengthpolymorphism (RFLP) to 24-locus variable-number tandem-repeat (VNTR) typing of Mycobacterium tuberculosis complex isolates in the national tuberculosis control program in The Netherlands, a

Full Text Available The aim of study was to identify the association of FSH beta-subunit gene polymorphisms with sperm quality traits. A total of 470 samples of normal mature bull from several breeds were used for population study and 127 bulls from National and Regional AI centre of Indonesia for association study. To amplify, a PCR-RFLP method was used and digested with Pst1 restriction enzyme. The allele frequency of the A and B in Bali cattle were (0.000 and (1.000, respectively. The absence of other allele A suggested that the Bali cattle was monomorphic, while Brahman, FH, Simmental and Limousine were polymorphic. The highest observed heterozygosity were found in Limousine (0.318 and the highest expected heterozygosity were in Simmental (0.420. The higher incident of percentage of sperm abnormalities were found in Simmental, Limousin, Brahman compared to Bali and FH. Among all types of sperm abnormalities, the abaxial and microcephalus were found in highest number.

Recent studies indicated that the aberrant gene expression of peroxiredoxin-6 (prdx6) was found in various kinds of cancers. Because of its biochemical function and gene expression pattern in cancer cells, the association between genetic polymorphism of Prdx6 and cancer onset is interesting. In this report, we have developed and implemented a…

Full Text Available Prompt and specific identification of fungemia agents is important in order to define clinical treatment. However, in most cases conventional culture identification can be considered to be time-consuming and not without errors. The aim of the present study was to identify the following fungemia agents: Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata, Cryptococcus neoformans, Cryptococcus gattii, and Histoplasma capsulatum using the polymerase chain reaction and restriction fragment lengthpolymorphism analysis (PCR/RFLP. More specifically: a to evaluate 3 different amplification regions, b to investigate 3 different restriction enzymes, and c to use the best PCR/RFLP procedure to indentify 60 fungemia agents from a culture collection. All 3 pairs of primers (ITS1/ITS4, NL4/ITS5 and Primer1/Primer2 were able to amplify DNA from the reference strains. However, the size of these PCR products did not permit the identification of all the species studied. Three restriction enzymes were used to digest the PCR products: HaeIII, Ddel and Bfal. Among the combinations of pairs of primers and restriction enzymes, only one (primer pair NL4/ITS5 and restriction enzyme Ddel produced a specific RFLP pattern for each microorganism studied. Sixty cultures of fungemia agents (selected from the culture collection of Fundação de Medicina Tropical do Amazonas - FMTAM were correctly identified by PCR/RFLP using the prime pair NL4/ITS5 and Ddel. We conclude that the method proved to be both simple and reproducible, and may offer potential advantages over phenotyping methods.

Changes that may have occurred over the past 50 years of hybrid breeding in maize (Zea maize L.) with respect to heterosis for yield and heterozygosity at the molecular level are of interest to both maize breeders and quantitative geneticists. The objectives of this study were twofold: The first, to compare two diallels produced from six older maize inbreds released in the 1950's and earlier and six newer inbreds released during the 1970's with respect to (a) genetic variation for restriction fragment lengthpolymorphisms (RFLPs) and (b) the size of heterosis and epistatic effects, and the second, to evaluate the usefulness of RFLP-based genetic distance measures in predicting heterosis and performance of single-cross hybrids. Five generations (parents, F1; F2, and backcrosses) from the 15 crosses in each diallel were evaluated for grain yield and yield components in four Iowa environments. Genetic effects were estimated from generation means by ordinary diallel analyses and by the Eberhart-Gardner model. Newer lines showed significantly greater yield for inbred generations than did older lines but smaller heterosis estimates. In most cases, estimates of additive x additive epistatic effects for yield and yield components were significantly positive for both groups of lines. RFLP analyses of inbred lines included two restriction enzymes and 82 genomic DNA clones distributed over the maize genome. Eighty-one clones revealed polymorphisms with at least one enzyme. In each set, about three different RFLP variants were typically found per RFLP locus. Genetic distances between inbred lines were estimated from RFLP data as Rogers' distance (RD), which was subdivided into general (GRD) and specific (SRD) Rogers' distances within each diallel. The mean and range of RDs were similar for the older and newer lines, suggesting that the level of heterozygosity at the molecular level had not changed. GRD explained about 50% of the variation among RD values in both sets. Cluster

Restriction fragment lengthpolymorphism (RFLP) is a difference in DNA sequences of organisms belonging to the same species. RFLPs are typically detected as DNA fragments of different lengths after digestion with various restriction endonucleases. The comparison of RFLPs allows investigators to analyze the frequency of occurrence of mutations, such as point mutations, deletions, insertions, and gross chromosomal rearrangements, in the progeny of stressed plants. The assay involves restriction enzyme digestion of DNA followed by hybridization of digested DNA using a radioactively or enzymatically labeled probe. Since DNA can be digested with methylation sensitive enzymes, the assay can also be used to analyze a methylation pattern of a particular locus. Here, we describe RFLP analysis using methylation-insensitive and methylation-sensitive enzymes.

A restriction fragment lengthpolymorphism (RFLP) map has been constructed of the nuclear genome of the plant pathogenic ascomycete Cochliobolus heterostrophus. The segregation of 128 RFLP and 4 phenotypic markers was analyzed among 91 random progeny of a single cross; linkages were detected among 126 of the markers. The intact chromosomal DNAs of the parents and certain progeny were separated using pulsed field gel electrophoresis and hybridized with probes used to detect the RFLPs. In this way, 125 markers were assigned to specific chromosomes and linkages among 120 of the markers were confirmed. These linkages totalled 941 centimorgans (cM). Several RFLPs and a reciprocal translocation were identified tightly linked to Tox1, a locus controlling host-specific virulence. Other differences in chromosome arrangement between the parents were also detected. Fourteen gaps of at least 40 cM were identified between linkage groups on the same chromosomes; the total map length was therefore estimated to be, at a minimum, 1501 cM. Fifteen A chromosomes ranging from about 1.3 megabases (Mb) to about 3.7 Mb were identified; one of the strains also has an apparent B chromosome. This chromosome appears to be completely dispensable; in some progeny, all of 15 markers that mapped to this chromosome were absent. The total genome size was estimated to be roughly 35 Mb. Based on these estimates of map length and physical genome size, the average kb/cM ratio in this cross was calculated to be approximately 23. This low ratio of physical to map distance should make this RFLP map a useful tool for cloning genes. PMID:1346261

Jul 11, 2011 ... Amplified fragment lengthpolymorphism (AFLP) technology was used to reveal the genetic variation in six species of Cycas collected from eleven natural populations. Two sets of primer with 4-selective nucleotides were used in this study and 78% polymorphism was found. The results correlated with.

The amplified restriction fragment lengthpolymorphism (AFLP) technique is a relatively new method for the analysis of polymorphism that has not yet been widely used in parasitology. In this article, Dan Masiga, Andy Tait and Mike Turner provide a brief introduction to AFLP and illustrate how it can be used in the investigation of marker inheritance in genetic crosses and in the analysis of polymorphism of field populations. They also briefly highlight the strengths and weaknesses of AFLP in comparison with other methods for detecting polymorphism and conclude that AFLP is a very useful addition to the range of techniques available.

New chicken Rfp-Y haplotypes were determined by the use of restriction fragment lengthpolymorphism (RFLP) and mixed lymphocyte culture (MLC) in four different chicken haplotypes, B15, B19, B21, B201. The RFLPpolymorphism was mapped to the Rfp-Y system by the use of a subclone (18.1) which maps...... near a polymorphic lectin gene located in the Rfp-Y system and DNA from families with known segregation of the implicated RFLPpolymorphism. For the first time it is shown that major histocompatibility complex class II genes in the Rfp-Y system have functional implications. Sequence information...

Infectious bursal disease (IBD), also known as Gumboro disease, is a highly contagious, immunosuppressive disease of immature chickens. It is caused by IBD virus (IBDV) and is responsible for major economic losses in the poultry industry worldwide. In this study, 280 bursa samples from 56 commercially reared chicken flocks in Turkey with clinical symptoms of IBD were examined for IBDVs using the reverse transcription (RT)-polymerase chain reaction (PCR)/restriction fragment lengthpolymorphism (RFLP) assay. The assay was conducted on a 743-bp fragment of the VP2 gene with the restriction enzymes BstNI, MboI, and SspI. The results indicate the existence of field isolates with new molecular patterns different from those previously published that may well be unique and specific to geographical regions.

Full Text Available Abstract Apolipoprotein AV (ApoAV gene variant, -1131T>C, is associated with increased triglyceride concentrations in all ethnic groups studied. An MseI based RFLP analysis is the most commonly used method for genotyping this SNP. We genotyped a large cohort comprising 1185 Asian Indians and 173 UK Caucasians for -1131T>C using an ARMS-PCR based tetra-primer method. For quality control, we re-genotyped approximately 10% random samples from this cohort utilizing the MseI RFLP, which showed a 2.9% (3/102 genotyping error rate between the two methods. To investigate further, we sequenced the 900 bp region around the -1131T>C polymorphism in 25 Asian Indians and 15 UK Caucasians and found a number of polymorphisms including the -987C>T polymorphism. Further analysis of the -987C>T SNP showed a higher rare allele frequency of 0.23 in Asian Indians (n = 158 compared to 0.09 in the UK Caucasians (n = 157. This SNP is located 4 bp from the 3' end of the RFLP forward primer and is in weak linkage disequilibrium with -1131T>C variant (r2 = 0.084 and D' = 1. Repeated RFLP analysis of seven subjects heterozygous for -987C>T (seven times, showed discordant results with the sequence at -1131T>C SNP nearly one third (15/49 of the time. We conclude that presence of -987C>T polymorphism in the forward primer of the MseI RFLP assay may lead to allelic drop-out and generate unforeseen errors in genotyping the -1131T>C polymorphism. Our results also emphasise the need for careful quality control in all molecular genetic studies, particularly while transferring genotyping methods between various ethnic groups.

DNA typing from forensic evidence is commonly used to identify individuals. However, when the quantity of the forensic evidence is insufficient, successful identification using DNA typing is impossible. Such evidence may also contain DNA from bacteria that occur naturally on the skin. In this study, we aimed to establish a profiling method using terminal restriction fragment lengthpolymorphisms (T-RFLPs) of the amplified bacterial 16S ribosomal RNA (rRNA) gene. First, the extraction and digestion processes were investigated, and the T-RFLP profiling method using the 16S rRNA gene amplicon was optimized. We then used this method to compare the profiles of bacterial flora from the hands of 12 different individuals. We found that the T-RFLP profiles from one person on different days displayed higher similarity than those between individuals. In a principal component analysis (PCA), T-RFLPs from each individual were closely clustered in 11 out of 12 cases. The clusters could be distinguished from each other, even when the samples were collected from different conditions. No major change of the profile was observed after six months except in two cases. When handprints on glass plates were compared, 11 of 12 individuals were assigned to a few clusters including the cluster corresponding to the correct individual. In conclusion, a method for reproducible T-RFLP profiling of bacteria from trace amounts of handprints was established. The profiles were obtained for particular individuals clustered in PCA and were experimentally separable from other individuals in most cases. This technique could provide useful information for narrowing down a suspect in a criminal investigation.

Full Text Available Context: Candida spp. is an emerging cause of bloodstream infections worldwide. Delay in speciation of Candida isolates by conventional methods and resistance to antifungal drugs in various Candida species are responsible for the increase in morbidity and mortality due to candidemia. Hence, the rapid identification of Candida isolates is very important for the proper management of patients with candidemia. Aims: The aim was to re-evaluate the identification of various Candida spp. by polymerase chain reaction (PCR-restriction fragment lengthpolymorphism (RFLP and to evaluate the accuracy, speed, and cost of phenotypic methodology versus PCR-RFLP. Settings and Design: Hospital-based cross-sectional study. Materials and Methods: Ninety consecutive clinical isolates of seven Candida species, isolated from blood of neonates and identified by routine phenotypic methods, were re-evaluated using universal primers internal transcribed spacer 1 (ITS1 and ITS4 for PCR amplification and Msp I restriction enzyme for RFLP. Statistical Analysis Used: Kappa test for agreement. Results: The results of PCR-RFLP were 100% in agreement with those obtained using conventional phenotypic methods. Identification could be achieved within 3 work days by both the methods. Our routine methods proved to be cost effective than PCR-RFLP. Conclusions: We can continue with our routine phenotypic methods and PCR-RFLP can be used for periodic quality control or when conventional methods fail to identify a species.

Coxiella burnetii, as an obligata intracellular bacterium, is the etiologic agent of Q-fever. It is widely distributed in nature and is responsible for infection in various animals (cattle, sheep, goat) and humans. C. burnetii has been isolated from milk, ticks and human patients with acute and chronic Q fever. Ticks are the principal vectors and reservoirs of C. burnetii. Since over 40 species of ticks have been found to be infected with C. burnetii, ticks can serve as indicators of infection in nature. In this study, total of 2472 ticks (1446 female, 1021 male and 5 nymphs) were collected from 38 provinces of Turkey. The ticks were gathered into groups of 1 to 7 ticks as to the provinces, species and gender for DNA extraction. Following DNA extraction, the groups were examined for the presence of C. burtii by using the CB1and CB2. The ticks collected from the province of Denizli (56 in total) were gathered into 13 groups according to the species and gender. From these groups, 6 were positive for C. burnetii. The ticks collected from Ankara province, total of 160 ticks, were grouped into 53 as to their species and gender, only one group was found to be positive for C. burnetii. The specificities of PCR products were evaluated by restriction analysis. The positive PCR products were digested with the enzyme Taq1 and for bands in order of 118, 57, 43 and 39 bp's were appeared such as seen in the positive control DNA (C. burnetii Nine Mile RSA493)

The pattern of inheritance of a Pst I restriction fragment lengthpolymorphism (RFLP) of the human placental alkaline phosphatase gene was studied in nine nuclear families by Southern blot hybridization analysis of genomic DNA. The dimorphic RFLP is defined by the presence of allelic fragments 1.0 kilobase and 0.8 kilobase long. The results of this study show that the two alleles of the Pst I RFLP of the placental alkaline phosphatase gene segregate as codominant traits according to Mendelian expectations. For a polymorphism to be useful as a genetic marker the probability that an offspring is informative (PIC) must be at least 0.15. The allelic frequency of the 1.0-kilobase allele is 0.21, which correlates to a probability that an offspring is informative of 0.275 and is indicative of a useful polymorphism. By using probes derived from different regions of the placental alkaline phosphatase cDNA, the mutated Pst I site causing the RFLP was located in the penultimate intron 2497 base pairs downstream from the transcriptional initiation site

A linkage map of distal mouse chromosome 1 was generated using restriction fragment lengthpolymorphism (RFLP) analysis of DNA prepared from 95 [C3H-gld/gld X Mus spretus)F1 X C3H-gld/gld] backcross mice. The gene order was: (centromere) C4bp, Ren-1,2, Ly-5, [At-3/gld], Apoa-2/Ly-17, Spna-1 (telomere). All mice expressing the phenotype of gld homozygotes were homozygous for the At-3 RFLP characteristic of C3H mice and none of the mice heterozygous for At-3 RFLPs had characteristics of gld homozygotes, demonstrating close linkage between these genes. The identification of an RFLP closely linked to the gld gene provides a starting point for the identification of a genetic defect that results in abnormal T cells and autoimmune disease. PMID:2894402

Recently, PCR-restriction fragment lengthpolymorphism (PCR-RFLP) of the urease genes of Helicobacter pylori was evaluated in a meta-analysis; acceptable discriminatory indices of the ureAB and C genes were found. In the present investigation, we found a discriminatory index of 0.95 for 191...... unrelated clinical H. pylori isolates with PCR-RFLP typing of the ureB gene (933 bp), combining the results obtained with restriction enzymes HaeIII and Sau3A, and a mixture of the enzymes. We therefore find that PCR-RFLP typing of the ureB gene of H. pylori with restriction enzymes HaeIII and Sau3A...

unrelated clinical H. pylori isolates with PCR-RFLP typing of the ureB gene (933 bp), combining the results obtained with restriction enzymes HaeIII and Sau3A, and a mixture of the enzymes. We therefore find that PCR-RFLP typing of the ureB gene of H. pylori with restriction enzymes HaeIII and Sau3A......Recently, PCR-restriction fragment lengthpolymorphism (PCR-RFLP) of the urease genes of Helicobacter pylori was evaluated in a meta-analysis; acceptable discriminatory indices of the ureAB and C genes were found. In the present investigation, we found a discriminatory index of 0.95 for 191...

Full Text Available Abstract Background Group A streptococcal (GAS infections can lead to the development of severe post-infectious sequelae, such as rheumatic fever (RF and rheumatic heart disease (RHD. RF and RHD are a major health concern in developing countries, and in indigenous populations of developed nations. The majority of GAS isolates are M protein-nontypeable (MNT by standard serotyping. However, GAS typing is a necessary tool in the epidemiologically analysis of GAS and provides useful information for vaccine development. Although DNA sequencing is the most conclusive method for M protein typing, this is not a feasible approach especially in developing countries. To overcome this problem, we have developed a polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP-based assay for molecular typing the M protein gene (emm of GAS. Results Using one pair of primers, 13 known GAS M types showed one to four bands of PCR products and after digestion with Alu I, they gave different RFLP patterns. Of 106 GAS isolates examined from the normal Thai population and from patients with GAS-associated complications including RHD, 95 isolates gave RFLP patterns that corresponded to the 13 known M types. Only 11 isolates gave RFLP patterns that differed from the 13 known M types. These were then analyzed by DNA sequencing and six additional M types were identified. In addition, we found that M93 GAS was the most common M type in the population studied, and is consistent with a previous study of Thai GAS isolates. Conclusion PCR-RFLP analysis has the potential for the rapid screening of different GAS M types and is therefore considerably advantageous as an alternative M typing approach in developing countries in which GAS is endemic.

Aims: This study was undertaken to investigate the usefulness of amplified fragment lengthpolymorphism (AFLP) in determining the population structure of Salmonella. Methods and Results: A total of 89 strains were subjected to AFLP analysis using the enzymes BglII and BspDI, a combination...

Internal transcribed spacer 2 region in 4 species of sandflies from China (Phlebotomus chinensis, Ph. wui, Ph. longiductus and Ph. alexandri) was genotyped by PCR-RFLP. Four kinds of RFLP band patterns were created by Dra I, that were A 450 bp, B 154 bp/127 bp/93 bp/66 bp/10 hp, C 311 bp/80 bp/60 bp, and D 353 bp/300 bp/160 bp/58 bp. There were identical pattern types in various sandflies, AA in Ph. chinensis, BB in Ph. alexandri, CC in Ph. wui, and DD in Ph. longiductus. The results indicated that PCR-RFLP can be used to identify the four species of Phlebotomus in China.

Mycobacterium tuberculosis, the etiologic agent of tuberculosis, causes large-scale morbidity and mortality, particularly in developing countries. In recent years, there has been a significant increase in the drug-resistant ability of M. tuberculosis, triggering a major public health crisis. A detailed analysis of the evolution of the mycobacterial genome helps to better understand the genotype-phenotype relationship in this bacterium. Different strain typing methods have already revealed the worldwide diversity of mycobacterial isolates. Therefore, DNA-fingerprinting tools have been developed to improve tuberculosis case detection and control. Molecular typing techniques allow to detect and follow the spread of individual strains of the M. tuberculosis complex (MTC), complementing conventional epidemiological methods. Among these techniques, restriction fragment lengthpolymorphism (RFLP) has been considered the standard method for genotyping of MTC. The aim of this work was to isolate M. tuberculosis from rodents in cattle farms contaminated with MTC located in the city of Booin-Zahra, Iran. A total of 100 samples were collected from the rodents in the contaminated farms and analyzed for the presence of Mycobacterium by growing the samples on Lowenstein-Jensen medium. All isolates were further identified by RFLP and DNA hybridization studies. As much as five samples showed the presence of Mycobacterium and these were subjected to PCR-16SrRNA, PCR-IS6110, and RD Typing (RD1, RD4, RD9, and RD12) methods. Further differentiation was performed with PvuII digestion (RFLP) and DNA hybridization using the polymorphic guanine/cytosine-rich repetitive sequences (PGRS) probe. The PGRS probe results classified two of the isolates as belonging to one cluster, whereas the remaining isolates were classified as belonging to different clusters. An analysis of the obtained genetic pattern and a comparison of these patterns with the genetic pattern of other infected farms allowed

ABSTRACT Spongospora subterranea f. sp. subterranea causes powdery scab in potatoes and is distributed worldwide. Genetic studies of this pathogen have been hampered due, in part, to its obligate parasitism and the lack of molecular markers for this pathogen. In this investigation, a single cystosorus inoculation technique was developed to produce large amounts of S. subterranea f. sp. subterranea plasmodia or zoosporangia in eastern black nightshade (Solanum ptycanthum) roots from which DNA was extracted. Cryopreservation of zoosporangia was used for long-term storage of the isolates. S. subterranea f. sp. subterranea-specific restriction fragment lengthpolymorphism (RFLP) markers were developed from randomly amplified polymorphic DNA (RAPD) fragments. Cystosori of S. subterranea f. sp. subterranea were used for RAPD assays and putative pathogen-specific RAPD fragments were cloned and sequenced. The fragments were screened for specificity by Southern hybridization and subsequent DNA sequence BLAST search. Four polymorphic S. subterranea f. sp. subterranea-specific probes containing repetitive elements, and one containing single copy DNA were identified. These RFLP probes were then used to analyze 24 single cystosorus isolates derived from eight geographic locations in the United States and Canada. Genetic variation was recorded among, but not within, geographic locations. Cluster analysis separated the isolates into two major groups: group I included isolates originating from western North America, with the exception of those from Colorado, and group II included isolates originating from eastern North America and from Colorado. The techniques developed in this study, i.e., production of single cystosorus isolates of S. subterranea f. sp. subterranea and development of RFLP markers for this pathogen, provide methods to further study the genetic structure of S. subterranea f. sp. subterranea.

Contamination with plastid small subunit (SSU) rDNA is a major drawback when analyzing the bacterial communities of plant roots using culture-independent methods. In this study, a polymerase chain reaction (PCR) primer, 783r, was designed and tested to specifically amplify the SSU rDNA of various bacterial species without amplifying the SSU rDNA of plant plastids. To confirm how useful the community analysis of rhizobacteria is using 783r, the terminal restriction fragment lengthpolymorphism (T-RFLP) method was performed with wheat (Triticum aestivum) and spinach (Spinacea oleracea) root samples. Using the standard T-RFLP method, a large T-RF peak of plant plastid SSU rDNA interfered with the bacterial community analysis. In contrast, the T-RFLP method using the 783r primer was able to detect the bacterial DNA while directly eliminating the influence of the plant-derived DNA extracted from the plant roots. Primer 783r might, therefore, be a useful PCR primer for the culture-independent analysis of bacterial communities in plant roots using SSU rDNA.

Full Text Available The combination of molecular and conventional epidemiological methods has improved the knowledge about the transmission of tuberculosis in urban populations. To examine transmission of tuberculosis in Havana, Cuba, with DNA fingerprinting, we studied 51 out of 92 Mycobacterium tuberculosis strains isolated from tuberculosis patients who resided in Havana and whose infection was culture-confirmed in the period from September 1997 to March 1998. Isolates from 28 patients (55% had unique IS6110 restriction fragment lengthpolymorphism (RFLP patterns, while isolates from 23 others (45% had identical patterns and belonged to 7 clusters. Three clusters consisting of six, five and two cases were each related to small outbreaks that occurred in a closed setting. Three other clustered cases were linked to a large outbreak that occurred in another institution. Younger patients were more correlated to clustering than older ones. The finding that 45% of the isolates had clustered RFLP patterns suggests that recent transmission is a key factor in the tuberculosis cases in Havana. The IS6110 RFLP typing made it possible to define the occurrence of outbreaks in two closed institutions.

The restriction fragment lengthpolymorphism (RFLP) of the class II HLA-DP subregion of the major histocompatibility complex (MHC) of humans has been unraveled by Southern blotting using DP/sub α/ and DP/sub β/ probes in a study of 46 unrelated individuals with known HLA-DP types. Contrary to earlier preliminary findings with a limited number of enzymes, the RFLP appears to be quite extensive both with the DP/sub β/ (14 different DNA markers defined by individual fragments or clusters thereof) and the DP/sub α/ (8 markers) probes, especially when enzyme recognizing only four base pairs were used. A few markers were absolutely or strongly associated with individual DP antigens, whereas most were associated with two or more DP antigens as defined by primed lymphocyte typing. Thus, Southern blotting seems feasible for typing for most DP determinants by specific fragments or subtraction between the various more broadly reactive DNA markers, and the RFLP provides further information on the DP subregion in addition to that provided by primed lymphocyte typing. In two recombinant families, the DP/sub β/ and DP/sub α/ DNA markers segregated with DP antigens, whereas the DR/sub β/, DQ/sub β/, DQ/sub α/, and DX/sub α/ markers followed the DR and DQ antigens

Full Text Available Restriction fragment lengthpolymorphism (RFLP analysis of mitochondrial DNA (mtDNA was employed to characterize species and populations of Astyanax, a Neotropical freshwater fish genus. Samples of five species, A. altiparanae, A. fasciatus, A. lacustris, A. scabripinnis paranae and A. schubarti, from the Upper Paraná and São Francisco river basins were analyzed. Two out of the ten restriction enzymes employed generated species-specific mtDNA patterns for each of the five species. MtDNA exhibited considerable polymorphism within and among populations. All populations sampled showed relatively high values of haplotype diversity. Geographically localized haplotypes were detected for A. altiparanae and A. fasciatus from the Upper Paraná and São Francisco basins. The relationships between populations are discussed.

Thermophilic campylobacters, particularly Campylobacter jejuni and C. coli are the main agents of human campylobacteriosis. Campylobacter contaminated chicken products is the most important source of foodborne gastroenteritis. Evaluation of genetic diversity among Campylobacter population is critical for understanding the epidemiology of this bacterium and developing effective control strategies against Campylobacter infections and other related disorders. The aim of this study was to investigate the polymorphism of thermophilic Campylobacter isolated from broiler fecal samples in Shiraz, southern Iran. Ninety Campylobacter isolates were recovered from broiler feces using enrichment process followed by cultivation method. The isolates were species typing on the basis of polymerase chain reaction (PCR) detection of 16SrRNA and multiplex PCR for determining two thermophilic species. To evaluate strain diversity of thermophilic Campylobacter isolates, flaA PCR-Restriction Fragment LengthPolymorphism (RFLP) was performed using DdeI restriction enzyme. All 90 Campylobacter isolates confirmed by m-PCR were successfully typed using flaA-PCR-RFLP. Eleven different types were defined according to flaA-typing method and the RFLP patterns were located at three separate clusters in RFLP image analysis dendrogram. Campylobacter jejuni isolates significantly showed more variety than C. coli isolates. A relatively low genetic diversity existed among C. jejuni and C. coli isolated from broilers in Shiraz, southern Iran. In our knowledge, this was the first report of genetic diversity among broiler originated human pathogen thermophilic campylobacters in Shiraz, southern Iran.

, furnished cage or aviary), were compared using Terminal Restriction Fragment LengthPolymorphism (T-RFLP). The distribution of genera in the microbiota in caecum was furthermore described by next generation sequencing of 16S rDNA libraries. RESULTS: Hens in the same cage type developed similar T-RFLP...

Similarity within bacterial populations is difficult to assess due to the limited number of characters available for evaluation and the heterogeneity of bacterial species. Currently, the preferred method used to evaluate the structure of bacterial populations is multilocus enzyme electrophoresis. However, this method is extremely cumbersome and only offers an indirect measure of genetic similarities. The development of a more direct and less cumbersome method for this purpose is warranted. Restriction fragment lengthpolymorphism analysis was evaluated as a tool for use in the study of bacterial population structures and in the epidemiology and surveillance of infectious disease. A collection of Neisseria meningitidis was available for use in the investigation of this technique. Neisseria meningitidis is the causative agent of epidemic cerebrospinal meningitis and septicemia as well as a variety of other clinical manifestations. Each isolate in the collection was defined in terms of serogroup specificity, clinical history, geographic source, and date of isolation. Forty -six strains were chosen for this study. The DNA from each strain was restricted with Pst1 and EcoR1 and electrophoresed on agarose gels. The DNA was transferred to nylon filters and hybridized with P ^{32} labeled DNA probes. Two randomly generated probes and a gene-specific probe were used to estimate the genetic similarities between and among the strains in the study population. A total of 28 different restriction fragment migration types were detected by the probes used. Data obtained from the RFLP analysis was analysed by cluster analysis and multivariate statistical methods. A total of 7 clones groups were detected. Two of these appear to be major clones that comprise 35% of the population. This analysis demonstrates the lack of structure within Neisseria meningitidis due primarily to a heterogenous population and the lack of geographic segregation. The potential utility of this technique as a

Terminal restriction fragment lengthpolymorphism (T-RFLP) analysis is a DNA-fingerprinting method that can be used for comparisons of the microbial community composition in a large number of samples. There is no consensus on how T-RFLP data should be treated and analyzed before comparisons between samples are made, and several different approaches have been proposed in the literature. The analysis of T-RFLP data can be cumbersome and time-consuming, and for large datasets manual data analysis is not feasible. The currently available tools for automated T-RFLP analysis, although valuable, offer little flexibility, and few, if any, options regarding what methods to use. To enable comparisons and combinations of different data treatment methods an analysis template and an extensive collection of macros for T-RFLP data analysis using Microsoft Excel were developed. The Tools for T-RFLP data analysis template provides procedures for the analysis of large T-RFLP datasets including application of a noise baseline threshold and setting of the analysis range, normalization and alignment of replicate profiles, generation of consensus profiles, normalization and alignment of consensus profiles and final analysis of the samples including calculation of association coefficients and diversity index. The procedures are designed so that in all analysis steps, from the initial preparation of the data to the final comparison of the samples, there are various different options available. The parameters regarding analysis range, noise baseline, T-RF alignment and generation of consensus profiles are all given by the user and several different methods are available for normalization of the T-RF profiles. In each step, the user can also choose to base the calculations on either peak height data or peak area data. The Tools for T-RFLP data analysis template enables an objective and flexible analysis of large T-RFLP datasets in a widely used spreadsheet application.

Amplified-fragment lengthpolymorphism (AFLP) is a whole-genome fingerprinting method based on selective amplification of restriction fragments. The potential of the method for the characterization of mycoplasmas was investigated in a total of 50 strains of human and animal origin, including......I restriction endonucleases and subsequent ligation of corresponding site-specific adapters. The amplification of AFLP templates with a single set of nonselective primers resulted in reproducible fingerprints of approximately 60 to 80 fragments in the size range of 50 to 500 bp, The method was able...

Candida sp. have been responsible for an increasing number of infections, especially in patients with immunodeficiency. Species-specific differentiation of Candida sp. is difficult in routine diagnosis. This identification can have a highly significant association in therapy and prophylaxis. This work has shown a new application of the terminal restriction fragment lengthpolymorphism (t-RFLP) method in the molecular identification of six species of Candida, which are the most common causes of fungal infections. Specific for fungi homocitrate synthase gene was chosen as a molecular target for amplification. The use of three restriction enzymes, DraI, RsaI, and BglII, for amplicon digestion can generate species-specific fluorescence labeled DNA fragment profiles, which can be used to determine the diagnostic algorithm. The designed method can be a cost-efficient high-throughput molecular technique for the identification of six clinically important Candida species.

Several yeast strains of the species Saccharomyces cerevisiae, S. bayanus and S. paradoxus, first identified by hybridization experiments and measurements of DNA/DNA homology, were characterized using polymerase chain reaction/restriction fragment lengthpolymorphism (PCR/RFLP) analysis of the MET2 gene. There was no exception to the agreement between this method and classical genetic analyses for any of the strains examined, so PCR/RFLP of the MET2 gene is a reliable and fast technique for delimiting S. cerevisiae and S. bayanus. Enological strains classified as S. bayanus, S. chevalieri, and S. capensis gave S. cerevisiae restriction patterns, whereas most S. uvarum strains belong to S. bayanus. Enologists should no longer use the name of S. bayanus for S. cerevisiae Gal strains, and should consider S. bayanus as a distinct species.

Full Text Available Snails of the genus Biomphalaria from Venezuela were subjected to morphological assessment as well as polymerase chain reaction and restriction fragment lengthpolymorphism (PCR-RFLP analysis. Morphological identification was carried out by comparison of characters of the shell and the male and female reproductive apparatus. The PCR-RFLP involved amplification of the internal spacer region ITS1 and ITS2 of the RNA ribosomal gene and subsequent digestion of this fragment by the restriction enzymes DdeI, MnlI, HaeIII and MspI. The planorbids were compared with snails of the same species and others reported from Venezuela and present in Brazil, Cuba and Mexico. All the enzymes showed a specific profile for each species, that of DdeI being the clearest. The snails were identified as B. glabrata, B. prona and B. kuhniana.

Susceptibility of the mosquito Aedes aegypti to the malarial parasite Plasmodium gallinaceum was investigated as a quantitative trait using restriction fragment lengthpolymorphisms (RFLP). Two F{sub 2} populations of mosquitoes were independently prepared from pairwise matings between a highly susceptible and a refractory strain of A. aegypti. RFLP were tested for association with oocyst development on the mosquito midgut. Two putative quantitative trait loci (QTL) were identified that significantly affect susceptibility. One QTL, pgs [2,LF98], is located on chromosome 2 and accounted for 65 and 49% of the observed phenotypic variance in the two populations, respectively. A second QTL, pgs[3,MalI], is located on chromosome 3 and accounted for 14 and 10% of the observed phenotypic variance in the two populations, respectively. Both QTL exhibit a partial dominance effect on susceptibility, wherein the dominance effect is derived from the refractory parent. No indication of epistasis between these QTL was detected. Evidence suggests that either a tightly linked cluster of independent genes or a single locus affecting susceptibility to various mosquito-borne parasites and pathogens has evolved near the LF98 locus; in addition to P. gallinaceum susceptibility, this general genome region has previously been implicated in susceptibility to the filaria nematode Brugia malayi and the yellow fever virus. 35 refs., 2 figs., 3 tabs.

Allozyme electrophoresis and restriction fragment lengthpolymorphism (RFLP) analyses were used to examine the genetic diversity of a collection of 18 Rhizobium leguminosarum bv. trifolii, 1 R. leguminosarum bv. viciae, and 2 R. meliloti strains. Allozyme analysis at 28 loci revealed 16 electrophoretic types. The mean genetic distance between electrophoretic types of R. leguminosarum and R. meliloti was 0.83. Within R. leguminosarum, the single strain of bv. viciae differed at an average of 0.65 from strains of bv. trifolii, while electrophoretic types of bv. trifolii differed at a range of 0.23 to 0.62. Analysis of RFLPs around two chromosomal DNA probes also delineated 16 unique RFLP patterns and yielded genetic diversity similar to that revealed by the allozyme data. Analysis of RFLPs around three Sym (symbiotic) plasmid-derived probes demonstrated that the Sym plasmids reflect genetic divergence similar to that of their bacterial hosts. The large genetic distances between many strains precluded reliable estimates of their genetic relationships. PMID:16348600

Immunocapture reverse transcriptase-polymerase chain reaction (RT-PCR) followed by restriction fragment lengthpolymorphism (RFLP) analysis of the product has been shown to be an effective procedure for discriminating serologically indistinguishable isolates of two plant viruses, raspberry bushy dwarf (RBDV) and zucchini yellow mosaic (ZYMV). For both viruses, only limited sequence information was available at the time of primer design, but most of the isolates which were tested could be amplified (the one exception being a serologically quite distinct isolate of ZYMV). Restriction endonucleases revealing diagnostic RFLPs were readily identified. Each of two isolates of ZYMV could be detected in the presence of the other and the relative proportions approximately quantified by visual estimation of the relative intensity of the appropriate bands. A range of isolates of different RBDV pathotypes were compared; isolates were grouped in ways that accorded with their known history. Computer analysis of the published sequence from which the primers had been derived showed the sequenced isolate to be identical with an isolate imported from the USSR. The PCR/RFLP procedure is rapid (it can be completed in less than 2 days), effective and will probably be generally applicable to distinguishing closely related virus isolates, even where little sequence information is available.

Developing novel strategies to increase the content of bioactive unsaturated fatty acids (FA) in ruminant-derived products requires a deeper understanding of rumen biohydrogenation and bacteria involved in this process. Although high-throughput pyrosequencing may allow for a great coverage of bacterial diversity, it has hardly been used to investigate the microbiology of ruminal FA metabolism. In this experiment, 454 pyrosequencing and a molecular fingerprinting technique (terminal restriction fragment lengthpolymorphism; T-RFLP) were used concurrently to assess the effect of diet supplementation with marine algae (MA) on the rumen bacterial community of dairy sheep. Eleven lactating ewes were divided in 2 lots and offered a total mixed ration based on alfalfa hay and concentrate (40:60), supplemented with 0 (control) or 8 (MA) g of MA/kg of dry matter. After 54 d on treatments, animals were slaughtered and samples of rumen content and fluid were collected separately for microbial analysis. Pyrosequencing yielded a greater coverage of bacterial diversity than T-RFLP and allowed the identification of low abundant populations. Conversely, both molecular approaches pointed to similar conclusions and showed that relevant changes due to MA addition were observed within the major ruminal phyla, namely Bacteroidetes, Firmicutes, and Proteobacteria. Decreases in the abundance of unclassified Bacteroidales, Porphyromonadaceae, and Ruminococcaceae and increases in as-yet uncultured species of the family Succinivibrionaceae, might be related to a potential role of these groups in different pathways of rumen FA metabolism. Diet supplementation with MA, however, had no effect on the relative abundance of Butyrivibrio and Pseudobutyrivibrio genera. In addition, results from both 454 pyrosequencing and T-RFLP indicate that the effect of MA was rather consistent in rumen content or fluid samples, despite inherent differences between these fractions in their bacterial composition

Full Text Available Blood flukes are among the most common disease causing pathogens infecting vertebrates, including humans and some of the world's most globally endangered fauna. Spirorchiid blood flukes are parasites of marine turtles, and are associated with pathology, strandings and mortalities worldwide. Their ova embolize in tissues and incite significant inflammatory responses, however attempts to draw correlations between species and lesions are frustrated by difficulties in identifying ova beyond the genus level. In this study, a newly developed terminal restriction fragment lengthpolymorphism (T-RFLP method was validated as a tool for differentiating between mixed spirorchiid ova in turtle tissue. Initially, a multiplex PCR was used to differentiate between the five genera of spirorchiid flukes. Following this, PCR was performed using genus/genera-specific fluorescently tagged primer pairs and PCR products digested analysis using restriction endonucleases. Using capillary electrophoresis, this T-RFLP method could differentiate between twelve species and genotypes of spirorchiid flukes in turtles. It was applied to 151 tissue samples and successfully identified the spirorchiid species present. It was found to be more sensitive than visual diagnosis, detecting infections in 28 of 32 tissues that were negative on histology. Spirorchiids were present in 96.7% of tissues tested, with Neospirorchis genotype 2 being the most prevalent, present in 93% of samples. Mixed infections were common, being present in 60.7% of samples tested. The method described here is, to our knowledge, the first use of the T-RFLP technique on host tissues or in an animal ecology context, and describes a significant advancement in the clinical capacity to diagnose a common cause of illness in our environment. It is proven as a sensitive, specific and cost-efficient means of identifying spirorchiid flukes and ova in turtles, with the potential to contribute valuable information to

Laboratory exercise is helpful for medical students to understand the basic principles of molecular biology and to learn about the practical applications of molecular biology. We have designed a lab course on molecular biology about the determination of single nucleotide polymorphism (SNP) in human REV3 gene, the product of which is a subunit of…

Full Text Available Canine atopic dermatitis (cAD is a common inflammatory skin disease that is considered to be a naturally occurring, spontaneous model of human atopic dermatitis (eczema. The aim of the paper was to identify of the SNP rs22114085 in different dog breeds. The material involved 52 dogs from 5 different breeds. Canine genomic DNA was isolated from saliva by modified method with using DNAzol® and linear polyacrylamide (LPA carrier and from blood by using commercial kit NucleospinBlood and used in order to estimate rs22114085 SNP genotypes by PCR-RFLP method. The PCR products were digested with DdeI restriction enzyme. The C allele was distributed in Czech Pointer, Chihuahua, German Wirehaired Pointer with an allele frequency ranging from 0.4545 to 1.00. In the population of Czech Pointer we detected all genotypes CC, CT and TT with frequency in male 0.25, 0.5833 and 0.1667, and in female 0.2728, 0.3636 and 0.3636, subsequently. In German Wirehaired Pointer was detected homozygote genotype CC in male and heterozygote genotype CT in female with frequency 1 and 1. In Chihuahua was observed homozygote genotype CC and heterozygote genotype CT with frequency 0.3333 and 0.6667, subsequently. In Golden retriever and Pincher we detected genotype TT with frequency 1.

In order to switch from IS6110 and polymorphic GC-rich repetitive sequence (PGRS) restriction fragment lengthpolymorphism (RFLP) to 24-locus variable-number tandem-repeat (VNTR) typing of Mycobacterium tuberculosis complex isolates in the national tuberculosis control program in The Netherlands, a detailed evaluation on discriminatory power and agreement with findings in a cluster investigation was performed on 3,975 tuberculosis cases during the period of 2004 to 2008. The level of discrimination of the two typing methods did not differ substantially: RFLP typing yielded 2,733 distinct patterns compared to 2,607 in VNTR typing. The global concordance, defined as isolates labeled unique or identically distributed in clusters by both methods, amounted to 78.5% (n = 3,123). Of the remaining 855 cases, 12% (n = 479) of the cases were clustered only by VNTR, 7.7% (n = 305) only by RFLP typing, and 1.8% (n = 71) revealed different cluster compositions in the two approaches. A cluster investigation was performed for 87% (n = 1,462) of the cases clustered by RFLP. For the 740 cases with confirmed or presumed epidemiological links, 92% were concordant with VNTR typing. In contrast, only 64% of the 722 cases without an epidemiological link but clustered by RFLP typing were also clustered by VNTR typing. We conclude that VNTR typing has a discriminatory power equal to IS6110 RFLP typing but is in better agreement with findings in a cluster investigation performed on an RFLP-clustering-based cluster investigation. Both aspects make VNTR typing a suitable method for tuberculosis surveillance systems. PMID:23363841

Monitoring the structure and dynamics of fungal communities in soils under agricultural and environmental disturbances is currently a challenge. In this study, a terminal restriction fragment lengthpolymorphism (T-RFLP) fingerprinting method was developed for the rapid comparison of fungal community structures. The terminal restriction fragment polymorphism of different regions of the small-subunit (SSU) ribosomal RNA (rRNA) gene was simulated by sequence comparison using 10 restriction enzymes, and analyzed among three different soils using fungal-specific primers. Polymerase chain reaction amplification of the 3' end of the SSU rRNA gene with the primer nu-SSU-0817-5' and with the fluorescently labelled primer nu-SSU-1536-3', and digestion of the amplicons with AluI and MboI were found to be optimal and were used in a standardized T-RFLP procedure. Both the number and the intensity of terminal restriction fragments detected by capillary gel electrophoresis were integrated in correspondence analyses. Three soils with contrasting physicochemical properties were differentiated according to the structure of their fungal communities. Assessment of the impact on the fungal community structure of the amendment of two soils with compost or manure confirmed the reproducibility and the sensitivity of the method. Shifts in the community structure were detected between non-amended and amended soil samples. In both soils, the shift differed with the organic amendment applied. In addition, the fungal community structures of the two soils were affected in a different way by the same organic amendment. The fingerprinting method provides a rapid tool to investigate the effect of various perturbations on the fungal communities in soils.

Similarity within bacterial populations is difficult to assess due to the limited number of characters available for evaluation and the heterogeneity of bacterial species. Currently, the preferred method used to evaluate the structure of bacterial populations is multilocus enzyme electrophoresis. However, this method is extremely cumbersome and only offers an indirect measure of genetic similarities. The development of a more direct and less cumbersome method for this purpose is warranted. Restriction fragment lengthpolymorphism analysis was evaluated as a tool for use in the study of bacterial population structures and in the epidemiology and surveillance of infectious disease. A collection of Neisseria meningitidis was available for use in the investigation of this technique. Neisseria meningitidis is the causative agent of epidemic cerebrospinal meningitis and septicemia as well as a variety of other clinical manifestations. Each isolate in the collection was defined in terms of serogroup specificity, clinical history, geographic source, and date of isolation. Forty-six strains were chosen for this study. The DNA from each strain was restricted with Pst1 and EcoR1 and electrophoresed on agarose gels. The DNA was transferred to nylon filters and hybridized with P 32 labeled DNA probes. Two randomly generated probes and a gene-specific probe were used to estimate the genetic similarities between and among the strains in the study population. A total of 28 different restriction fragment migration types were detected by the probes used. Data obtained from the RFLP analysis was analyzed by cluster analysis and multivariate statistical methods. A total of 7 clones groups were detected. Two of these appear to be major clones that comprise 35% of the population

Similarity within bacterial populations is difficult to assess due to the limited number of characters available for evaluation and the heterogeneity of bacterial species. Currently, the preferred method used to evaluate the structure of bacterial populations is multilocus enzyme electrophoresis. However, this method is extremely cumbersome and only offers an indirect measure of genetic similarities. The development of a more direct and less cumbersome method for this purpose is warranted. Restriction fragment lengthpolymorphism analysis was evaluated as a tool for use in the study of bacterial population structures and in the epidemiology and surveillance of infectious disease. A collection of Neisseria meningitidis was available for use in the investigation of this technique. Neisseria meningitidis is the causative agent of epidemic cerebrospinal meningitis and septicemia as well as a variety of other clinical manifestations. Each isolate in the collection was defined in terms of serogroup specificity, clinical history, geographic source, and date of isolation. Forty-six strains were chosen for this study. The DNA from each strain was restricted with Pst1 and EcoR1 and electrophoresed on agarose gels. The DNA was transferred to nylon filters and hybridized with P{sup 32} labeled DNA probes. Two randomly generated probes and a gene-specific probe were used to estimate the genetic similarities between and among the strains in the study population. A total of 28 different restriction fragment migration types were detected by the probes used. Data obtained from the RFLP analysis was analyzed by cluster analysis and multivariate statistical methods. A total of 7 clones groups were detected. Two of these appear to be major clones that comprise 35% of the population.

The taxonomic validity of morphological group III Acanthamoeba spp. is uncertain. In the present study, six type strains of group III Acanthamoeba spp., A. culbertsoni, A. healyi, A. pustulosa, A. palestinensis, A. royreba and A. lenticulata were subjected for the evaluation of their taxonomic validity by comparison of the isoenzyme patterns by isoelectic focusing on polyacrylamide gels, mitochondrial DNA (Mt DNA) restriction fragment lengthpolymorphism (RFLP), and small subunit ribosomal DNA (ssu rDNA) PCR-RFLP patterns. The Mt DNA RFLP patterns were heterogeneous between the species. The type strains of A. palestinensis and A. pustulosa showed almost identical patterns of isoenzymes and rDNA PCR RFLP with an estimated sequence divergence of 2.6%. The other species showed heterogeneous patterns of isoenzymes and rDNA PCR-RFLP. It is likely that A. pustulosa is closely related with A. palestinensis and that the former may be regarded as a junior synonym of the latter.

Full Text Available Objectives: The aim of this investigation was to simultaneously detect and differentiate Mycoplasma genitalium and Ureaplasma urealyticum in female patients suffering from genital complications by polymerase chain reaction (PCR-restriction fragment lengthpolymorphism (RFLP. Materials and Methods : Genital swabs were taken from 210 patients. They were transported to the laboratory in phosphate-buffered saline. For PCR, samples were analysed with genus-specific MyUu-R and MyUu-F primers. This primer set, which was originally designed in our laboratory, amplified a 465 bp fragment (M. genitalium and a 559 bp fragment (U. urealyticum. Samples containing a band of the expected sizes for the Mycoplasma strains were subjected to digestion with a restriction endonuclease enzyme of TaqI and Cac8I. Results: Of the 210 samples, a total of 100 (47.6% samples were found to be positive for Mycoplasmas (seven M. genitalium isolates, 3.3%; and 89 U. urealyticum isolates, 42.4%, and coinfections with both species were detected in four samples (1.9%. The PCR-RFLP results showed that M. genitalium and U. urealyticum are different by enzyme patterns. Conclusion: PCR-RFLP offers a rapid and easily applicable protocol to simultaneous detection and differentiation of M. genitalium and U. urealyticum from clinical samples when specific primers and restriction enzymes are used.

Wider availability but lack of legal market trades has given feline meat a high potential for use as an adulterant in common meat and meat products. However, mixing of feline meat or its derivatives in food is a sensitive issue, since it is a taboo in most countries and prohibited in certain religions such as Islam and Judaism. Cat meat also has potential for contamination with of severe acute respiratory syndrome, anthrax and hepatitis, and its consumption might lead to an allergic reaction. We developed a very short-amplicon-length (69 bp) PCR assay, authenticated the amplified PCR products by AluI-restriction digestion followed by its separation and detection on a lab-on-a-chip-based automated electrophoretic system, and proved its superiority over the existing long-amplicon-based assays. Although it has been assumed that longer DNA targets are susceptible to breakdown under compromised states, scientific evidence for this hypothesis has been rarely documented. Strong evidence showed that shorter targets are more stable than the longer ones. We confirmed feline-specificity by cross-challenging the primers against 10 different species of terrestrial, aquatic and plant origins in the presence of a 141-bp site of an 18S rRNA gene as a universal eukaryotic control. RFLP analysis separated 43- and 26-bp fragments of AluI-digest in both the gel-image and electropherograms, confirming the original products. The tested detection limit was 0.01% (w/w) feline meat in binary and ternary admixed as well as meatball matrices. Shorter target, better stability and higher sensitivity mean such an assay would be valid for feline identification even in degraded specimens.

Molecular methods incorporating nested PCR-restriction fragment lengthpolymorphism (RFLP) analysis of the 18S rRNA gene of Cryptosporidium species were validated to assess performance based on limit of detection (LoD) and for detecting and resolving mixtures of species and genotypes within a single sample. The 95% LoD was determined for seven species (Cryptosporidium hominis, C. parvum, C. felis, C. meleagridis, C. ubiquitum, C. muris, and C. andersoni) and ranged from 7 to 11 plasmid template copies with overlapping 95% confidence limits. The LoD values for genomic DNA from oocysts on microscope slides were 7 and 10 template copies for C. andersoni and C. parvum, respectively. The repetitive nested PCR-RFLP slide protocol had an LoD of 4 oocysts per slide. When templates of two species were mixed in equal ratios in the nested PCR-RFLP reaction mixture, there was no amplification bias toward one species over another. At high ratios of template mixtures (>1:10), there was a reduction or loss of detection of the less abundant species by RFLP analysis, most likely due to heteroduplex formation in the later cycles of the PCR. Replicate nested PCR was successful at resolving many mixtures of Cryptosporidium at template concentrations near or below the LoD. The cloning of nested PCR products resulted in 17% of the cloned sequences being recombinants of the two original templates. Limiting-dilution nested PCR followed by the sequencing of PCR products resulted in no sequence anomalies, suggesting that this method is an effective and accurate way to study the species diversity of Cryptosporidium, particularly for environmental water samples, in which mixtures of parasites are common. PMID:21498746

The aim of this work was to sequence the entirecoding region of ACACA gene in Valle del Belice sheep breed to identify polymorphic sites. A total of 51 coding exons of ACACA gene were sequenced in 32 individuals of Valle del Belice sheep breed. Sequencing analysis and alignment of obtained sequences showed the ...

Full Text Available The escalating occurrence of cyanobacterial toxic blooms demands a better understanding of genetic variability as an auxiliary expedient in species identification, collaborating with the monitoring of water destined to public supply. This study aimed at the unraveling of genetic polymorphism in the toxic and nontoxic strains of Microcystis (Cyanobacteria species, isolated from diverse Brazilian localities through the RFLP-PCR technique applied to the c-phycocyanin encoding operon and its intergenic spacer (cpcBA-IGS. Eighteen strains belonging to M. aeruginosa, M. panniformis, M. protocystis and M. wesenbergii, plus two other unidentified strains, were analyzed by means of the morphological and molecular data. The molecular data constituted three groups with low similarity values unrelated to the geographical origin, toxicity or morphospecies. A high genetic variability among the studied populations was unveiled by the results. Brazilian populations of Microcystis spp. displayed high genetic diversity when compared to those from Australia, Japan, United States and Europe. This ample genetic diversity could be observed through the diverse eletrophoretic profiles obtained among the strains from a single species. The presence of toxic and non-toxic strains was observed in the same species, as M. aeruginosa.A ocorrência de florações de cianobactérias tóxicas demanda um melhor entendimento da variabilidade genética como um instrumento auxiliar na identificação de espécies colaborando, assim, com o monitoramento de águas destinadas ao abastecimento público. Este estudo objetivou o conhecimento do polimorfismo genético de linhagens tóxicas e não tóxicas de espécies de Microcystis (Cyanobacteria, isoladas de diversas localidades brasileiras, utilizando a técnica molecular RFLP-PCR para o operon que codifica para a c-ficocianina e seu espaçador intergênico (cpcBA-IGS. Foram analisadas dezoito linhagens pertencentes as esp

The aim of the study was to determine by Polymerase Chain Reaction-Restriction Fragment LengthPolymorphism (PCR-RFLP) method the gene and genotype frequencies of PRL gene in native East Anatolian Red (EAR) cattle, which are raised as a genetic resource in Turkey. PCR-RFLP analysis involved the use of the ...

Evaluation of effectiveness of restriction fragment lengthpolymorphism (RFLP) analysis of the 16S rRNA gene of microorganisms injected into an oil reservoir, for monitoring their levels over time, was conducted. Two microorganisms, enterobacter cloacae TRC-322 and Bacillus licheniformis TRC-18-2-a, were focused in this paper among the microorganisms selected for injection, and gene fragments of the 16S rRNA gene of these microorganisms were amplified by polymerase chain reaction (PCP), using one set of universal primers. Samples of the reservoir brine and reservoir rock were obtained; the microorganisms inhabiting in the reservoir were isolated from these samples, and the 16S rRNA gene of these microorganisms was amplified, condition remaining the same. RFLP analysis was performed on the 16S rRNA gene of each of these microorganisms, using restriction endonucleases HhaI, MspI, AluI and TaqI as necessary. Comparison of the resultant rRNA gene fragments, demonstrated that closely-related species displaying RFLP profile similar to that of E. cloacae TRC-322 or B. licheniformis TRC-18-2-a were not among the microorganisms isolated from the reservoir. PCR-RFLP analysis of the 16S rRNA gene, using the protocol; presented in this paper, is effective to detect the presence appropriate injecting microorganisms. This method was also effective for studying microorganisms isolated from the reservoir, which have the ability to grow on a molasses. (author)

Objectives: In this study we have evaluated the genotoxic potential of pesticides acephate and profenofos by polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP) assay with the mosquito Culex quinquefasciatus taken as experimental model. Material and Methods: Second instar larvae were treated with LC20 of each pesticide for 24 h and induced mutations in the sequence of mitochondrial 16S rRNA gene were studied from restriction patterns generated with PacI and PsiI rest...

A simple and robust biochemistry laboratory experiment is described that uses restriction fragment lengthpolymorphism (RFLP) of polymerase chain reaction (PCR) products to verify the identity of a potentially valuable horse. During the first laboratory period, students purify DNA from equine samples and amplify two loci of mitochondrial DNA. During the second laboratory period, students digest PCR products with restriction enzymes and analyze the fragment sizes through agarose gel electropho...

A total of 17 isolates of Colletotrichum from alfalfa (Medicago sativa L.) and red clover (Trifolium pratense L.) plants with anthracnose symptoms were collected from 11 districts in Serbia during 2005-2010 and tested for variability in vegetative compatibility groups (VCGs) and restriction fragment lengthpolymorphisms (RFLP). Nitrate nonutilising (nit) mutants were isolated from each of investigated C. destructivum isolates by selecting chlorate-resistant...

Phylogenetic relationships among 19 Musa species or cultivars were examined through DNA fingerprinting with amplified fragment lengthpolymorphism (AFLP) analysis. The AFLP analysis was performed on the Musa species or cultivars with 21 primer combinations, yielding a total of 6,348 DNA bands, among which 6,113 (96.3%) were polymorphic. M. itinerans var. formosana demonstrated 133 monomorphic bands, which is the most among all samples. Unweighted pair–group method with arithmetic averages was...

Amplified fragment lengthpolymorphism (AFLP) is a new molecular marker to detect genomic polymorphism. This new technology has advantages of high resolution, good stability, and reproducibility. Great achievements have been derived in recent years in AFLP related technologies with several AFLP expanded methodologies available. AFLP technology has been widely used in the fields of plant, animal, and microbes. It has become one of the hotspots in Forensic Botany. This review focuses on the recent advances of AFLP and its applications in forensic biology.

The genetic diversity among a worldwide collection of 120 strains of Ralstonia solanacearum was assessed by restriction fragment lengthpolymorphism (RFLP) analysis of amplified fragments from the hrp gene region. Five amplified fragments appeared to be specific to R. solanacearum. Fifteen different profiles were identified among the 120 bacterial strains, and a hierarchical cluster analysis distributed them into eight clusters. Each cluster included strains belonging to a single biovar, except for strains of biovars 3 and 4, which could not be separated. However, the biovar 1 strains showed rather extensive diversity since they were distributed into five clusters whereas the biovar 2 and the biovar 3 and 4 strains were gathered into one and two clusters, respectively. PCR-RFLP analysis of the hrp gene region confirmed the results of previous studies which split the species into an “Americanum” division including biovar 1 and 2 strains and an “Asiaticum” division including biovar 3 and 4 strains. However, the present study showed that most of the biovar 1 strains, originating from African countries (Reunion Island, Madagascar, Zimbabwe, and Angola) and being included in a separate cluster, belong to the “Asiaticum” rather than to the “Americanum” division. These African strains could thus have evolved separately from other biovar 1 strains originating from the Americas. PMID:10224018

An analysis of the dietary content of haematophagous insects can provide important information about the transmission networks of certain zoonoses. The present study evaluated the potential of polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome B (cytb) gene to differentiate between vertebrate species that were identified as possible sources of sandfly meals. The complete cytb gene sequences of 11 vertebrate species available in the National Center for Biotechnology Information database were digested with Aci I, Alu I, Hae III and Rsa I restriction enzymes in silico using Restriction Mapper software. The cytb gene fragment (358 bp) was amplified from tissue samples of vertebrate species and the dietary contents of sandflies and digested with restriction enzymes. Vertebrate species presented a restriction fragment profile that differed from that of other species, with the exception of Canis familiaris and Cerdocyon thous. The 358 bp fragment was identified in 76 sandflies. Of these, 10 were evaluated using the restriction enzymes and the food sources were predicted for four: Homo sapiens (1), Bos taurus (1) and Equus caballus (2). Thus, the PCR-RFLP technique could be a potential method for identifying the food sources of arthropods. However, some points must be clarified regarding the applicability of the method, such as the extent of DNA degradation through intestinal digestion, the potential for multiple sources of blood meals and the need for greater knowledge regarding intraspecific variations in mtDNA.

Full Text Available An analysis of the dietary content of haematophagous insects can provide important information about the transmission networks of certain zoonoses. The present study evaluated the potential of polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP analysis of the mitochondrial cytochrome B (cytb gene to differentiate between vertebrate species that were identified as possible sources of sandfly meals. The complete cytb gene sequences of 11 vertebrate species available in the National Center for Biotechnology Information database were digested with Aci I, Alu I, Hae III and Rsa I restriction enzymes in silico using Restriction Mapper software. The cytb gene fragment (358 bp was amplified from tissue samples of vertebrate species and the dietary contents of sandflies and digested with restriction enzymes. Vertebrate species presented a restriction fragment profile that differed from that of other species, with the exception of Canis familiaris and Cerdocyon thous. The 358 bp fragment was identified in 76 sandflies. Of these, 10 were evaluated using the restriction enzymes and the food sources were predicted for four: Homo sapiens (1, Bos taurus (1 and Equus caballus (2. Thus, the PCR-RFLP technique could be a potential method for identifying the food sources of arthropods. However, some points must be clarified regarding the applicability of the method, such as the extent of DNA degradation through intestinal digestion, the potential for multiple sources of blood meals and the need for greater knowledge regarding intraspecific variations in mtDNA.

Objective was to verify the incidence of cryptosporidiosis among Turkish elementary school students. The study was conducted in the Dept. of Parasitology, Faculty of Medicine, Ege University, Turkey during a 3-month period in 2006. We assessed the fecal samples of 707 children using modified acid-fast and phenol-auramine staining followed by modified Ritchie concentration method. All cryptosporidium species isolates were analysed by nested polymerase chain reaction (PCR) and restriction fragment lengthpolymorphism (RFLP) to differentiate genotypes of the isolates. After the coprological examination, 4 samples were found to be positive for cryptosporidium species oocysts. In the present study, all 4 oocysts were of zoonotic origin and belonged to cryptoporodium parvum genotype 2 indicating that in Turkey the potential sources of human cryptosporidiosis is from animals. The application of genotyping to clinical isolates of cryptosporidium has significantly increased our knowledge and understanding of the distribution and epidemiology of this parasite. The PCR and RFLP techniques represent a more rapid and simple method of genotyping to support epidemiological and clinical investigations than conventional analytical DNA techniques. (author)

Full Text Available Triatoma dimidiata is the most important Chagas disease insect vector in Central America as this species is primarily responsible for Trypanosoma cruzi transmission to humans, the protozoan parasite that causes Chagas disease. T. dimidiata sensu lato is a genetically diverse assemblage of taxa and effective vector control requires a clear understanding of the geographic distribution and epidemiological importance of its taxa. The nuclear ribosomal internal transcribed spacer 2 (ITS-2 is frequently used to infer the systematics of triatomines. However, oftentimes amplification and sequencing of ITS-2 fails, likely due to both the large polymerase chain reaction (PCR product and polymerase slippage near the 5' end. To overcome these challenges we have designed new primers that amplify only the 3'-most 200 base pairs of ITS-2. This region distinguishes the ITS-2 group for 100% of known T. dimidiata haplotypes. Furthermore, we have developed a PCR-restriction fragment lengthpolymorphism (RFLP approach to determine the ITS-2 group, greatly reducing, but not eliminating, the number of amplified products that need to be sequenced. Although there are limitations with this new PCR-RFLP approach, its use will help with understanding the geographic distribution of T. dimidiata taxa and can facilitate other studies characterising the taxa, e.g. their ecology, evolution and epidemiological importance, thus improving vector control.

SUMMARY. This study aimed to genotype infectious bursal disease virus (IBDV) isolates from the Minas Gerais state poultry industry. RNA was extracted from bursae obtained from field cases without passage or commercial vaccines. Genetic subtyping of IBDV isolates and vaccine strains was carried out by the reverse transcriptase-polymerase chain reaction (RT-PCR) and restriction fragment lengthpolymorphism (RFLP) analysis. A 588-bp fragment in the VP1 gene, an 847-bp fragment in the VP2 gene, and a 320-bp fragment in the VP3 gene were amplified by PCR and digested with restriction enzymes PstI and ScaI (VP1); BamHI, BstEII, and PstI (VP2); and NcoI, ScaI, and XbaI (VP3). Our work shows that complementing the clinical history of the outbreaks with RT-PCR followed by RFLP analysis using PstI for VP1, BamHI for VP2, and XbaI for VP3 allowed an accurate classification of a causative agent as a very virulent IBDV.

Full Text Available Aim: We aimed to identify the distribution of species in candidal strains isolated from clinical samples and restriction fragment lengthpolymorphism (RFLP method based on Msp I and Bln I restrictive enzyme cuts of polymerase chain reaction (PCR products after the amplification of ITS1 and ITS2 regions of rDNA genotypically. Materials and Methods: One hundred and fifty candidal strains isolated from various clinical samples were studies/ included. Phenotypic species assessment was performed using automated VITEK-2 system and kit used with the biochemical tests. Common genomic region amplification peculiar to candidal strains was carried out using ITS1 and ITS2 primer pairs. After the amplification, PCR products were cut with Msp I and Bln I restriction enzymes for species identification. Results: The majority of Candida isolates were isolated from urine (78.6% while other isolates were composed of strains isolated from swab, wound, blood and other samples by 11.3%, 3.3%, 2% and 4.7%, respectively. The result of RFLP analysis carried out with Msp I and Bln I restriction enzymes showed that candidal strains were Candida albicans by 45.3%, Candida glabrata by 19.3%, Candida tropicalis by 14.6%, Candida parapsilosis by 5.3%, Candida krusei by 5.3%, Candida lusitaniae by 0.6% and other candidal strains by 9.3%. Conclusion: When the ability to identify Candida to species level of phenotypic and PCR-RFLP methods was assessed, a great difference was found between these two methods. It may be argued that Msp I and Bln I restriction enzyme fragments can be used in the identification of medically important Candida species. Further studies are needed to develop this kind of restriction profile to be used in the identification of candidal strains.

A molecular method based on PCR-restriction fragment lengthpolymorphism (RFLP) analysis of internal transcribed spacer (ITS) ribosomal DNA sequences was designed to rapidly identify fungal species, with members of the genus Pleurotus as an example. Based on the results of phylogenetic analysis of ITS sequences from Pleurotus, a PCR-RFLP endonuclease autoscreening (PRE Auto) program was developed to screen restriction endonucleases for discriminating multiple sequences from different species. The PRE Auto program analyzes the endonuclease recognition sites and calculates the sizes of the fragments in the sequences that are imported into the program in groups according to species recognition. Every restriction endonuclease is scored through the calculation of the average coefficient for the sequence groups and the average coefficient for the sequences within a group, and then virtual electrophoresis maps for the selected restriction enzymes, based on the results of the scoring system, are displayed for the rapid determination of the candidate endonucleases. A total of 85 haplotypes representing 151 ITS sequences were used for the analysis, and 2,992 restriction endonucleases were screened to find the candidates for the identification of species. This method was verified by an experiment with 28 samples representing 12 species of Pleurotus. The results of the digestion by the restriction enzymes showed the same patterns of DNA fragments anticipated by the PRE Auto program, apart from those for four misidentified samples. ITS sequences from 14 samples (of which nine sequences were obtained in this study), including four originally misidentified samples, confirmed the species identities revealed by the PCR-RFLP analysis. The method developed here can be used for the identification of species of other living microorganisms.

Terminal restriction fragment lengthpolymorphism (T-RFLP) can be used to assess how land use management changes the dominant members of bacterial communities. We compared T-RFLP profiles obtained via amplification with forward primers (27, 63F) each coupled with the fluorescently labeled reverse primer (1392R) and multiple restriction enzymes to determine the best combination for interrogating soil bacterial populations in an agricultural soil used for potato production. Both primer pairs provide nearly universal recognition of a 1,400-bp sequence of the bacterial domain in the V(1)-V(3) region of the 16S ribosomal RNA (rRNA) gene relative to known sequences. Labeling the reverse primer allowed for direct comparison of each forward primer and the terminal restriction fragments' relative migration units obtained with each primer pair and restriction enzyme. Redundancy analysis (RDA) and nested multivariate analysis of variance (MANOVA) were used to assess the effects of primer pair and choice of restriction enzyme on the measured relative migration units. Our research indicates that the 63F-1392R amplimer pair provides a more complete description with respect to the bacterial communities present in this potato (Solanum tuberosum L.)-barley (Hordeum vulgare L.) rotation over seeded to crimson clover (Trifolium praense L.). Domain-specific 16S rRNA gene primers are rigorously tested to determine their ability to amplify across a target region of the gene. Yet, variability within or between T-RFLP profiles can result from factors independent of the primer pair. Therefore, researchers should use RDA and MANOVA analyses to evaluate the effects that additional laboratory and environmental variables have on bacterial diversity.

At least 13 characterized Leishmania species are known to infect humans in South America. Five of these parasites are transmitted in the sylvatic ecotopes of the whole French Guianan territory and responsible for cutaneous leishmaniasis. For the diagnosis of cutaneous leishmaniasis, restriction fragment lengthpolymorphism (RFLP) analyses have shown promising results. Thus, the end of the small subunit and internal transcribed spacer 1 of the rRNA genes were sequenced and targeted by PCR-RFLP analysis in the 10 main New World (NW) Leishmania species from the two subgenera. Then, the procedure was tested on 40 samples from patients with cutaneous leishmaniasis, and its results were compared with those of conventional methods. (i) The results of this simple genus-specific method were in agreement with those of previous isoenzyme analyses. (ii) This method distinguished the most medically relevant Leishmania species with only one enzyme (RsaI). (iii) This method could be performed directly on human biopsy specimens (sensitivity of 85.7%). Performing NW Leishmania species typing rapidly and easily in the field constitutes a very valuable improvement for detection of Leishmania spp. Revealing great diversity with several enzymes, this method could also be useful for taxonomic, ecological, and epidemiological studies in space and time.

Full Text Available Abstract Background Expansion of polyglutamine-encoding CAG trinucleotide repeats has been identified as the pathogenic mutation in nine different genes associated with neurodegenerative disorders. The majority of individuals clinically diagnosed with spinocerebellar ataxia do not have mutations within known disease genes, and it is likely that additional ataxias or Huntington disease-like disorders will be found to be caused by this common mutational mechanism. We set out to determine the length distributions of CAG-polyglutamine tracts for the entire human genome in a set of healthy individuals in order to characterize the nature of polyglutamine repeat length variation across the human genome, to establish the background against which pathogenic repeat expansions can be detected, and to prioritize candidate genes for repeat expansion disorders. Results We found that repeats, including those in known disease genes, have unique distributions of glutamine tract lengths, as measured by fragment analysis of PCR-amplified repeat regions. This emphasizes the need to characterize each distribution and avoid making generalizations between loci. The best predictors of known disease genes were occurrence of a long CAG-tract uninterrupted by CAA codons in their reference genome sequence, and high glutamine tract length variance in the normal population. We used these parameters to identify eight priority candidate genes for polyglutamine expansion disorders. Twelve CAG-polyglutamine repeats were invariant and these can likely be excluded as candidates. We outline some confusion in the literature about this type of data, difficulties in comparing such data between publications, and its application to studies of disease prevalence in different populations. Analysis of Gene Ontology-based functions of CAG-polyglutamine-containing genes provided a visual framework for interpretation of these genes' functions. All nine known disease genes were involved in DNA

The functional role of the exon 3 growth hormone receptor (d3GHR) polymorphism in human and its distributions in different populations is not clearly understood. The presence of full length growth hormone (flGHR) is the most important in metabolic risk factors. The aim of this study was to define the frequency distribution of d3GHR/full-length GHR in an Iranian population. The presence of the d3GHR polymorphism in healthy volunteers blood DNA (n=80, male=30 and female=50) was assessed by PCR ...

Associations were analysed between polymorphisms of the growth hormone gene (GH-MspI) (localized in intron 3) and milk production traits of Beijing Holstein cows (a total of 543 cows). Polymerase chain reaction (PCR)-restriction fragment lengthpolymorphism (RFLP) method was used for identification of various ...

The objective of this present study was to investigate relationships between the growth hormone gene restriction fragment lengthpolymorphism (RFLP) and bull sperm characteristics. A total of 89 bulls from two semen evaluation stations were genotyped for the bovine growth hormone (bGH)-AluI polymorphism by ...

The aim of this study was to detect the genetic polymorphism of GH gene in five camel breeds reared in Egypt which are Sudany, Somali, Mowaled, Maghrabi and Falahy, using polymerase chain reaction- restriction fragment lengthpolymorphism (PCR-RFLP) technique. Also, this work aimed to identify the single nucleotide ...

Aug 14, 2012 ... (Islam and Shepherd, 1992). But these markers were not considered suitable for large scale mapping. With the recent introduction of molecular biology, DNA based markers including polymerase chain reaction (PCR), restriction fragment lengthpolymorphism (RFLP), single nucleotide polymorphisms ...

The objective of this study was the detection of the restriction fragment lengthpolymorphism (RFLP) and single nucleotide polymorphisms (SNPs) of KISS1 gene in six major Egyptian small ruminant breeds. The primers used in this study flanked a 377 bp fragment from intron 1 of KISS1 gene in sheep and goat. These PCR ...

Background There is a likely genetic component to transsexualism, and genes involved in sex steroidogenesis are good candidates. We explored the specific hypothesis that male-to-female transsexualism is associated with gene variants responsible for undermasculinization and/or feminization. Specifically, we assessed the role of disease-associated repeat lengthpolymorphisms in the androgen receptor (AR), estrogen receptor β (ERβ), and aromatase (CYP19) genes. Methods Subject-control analysis included 112 male-to-female transsexuals and 258 non-transsexual males. Associations and interactions were investigated between CAG repeat length in the AR gene, CA repeat length in the ERβ gene, and TTTA repeat length in the CYP19 gene and male-to-female transsexualism. Results A significant association was identified between transsexualism and the AR allele, with transsexuals having longer AR repeat lengths than non-transsexual male control subjects (p = .04). No associations for transsexualism were evident in repeat lengths for CYP19 or ERβ genes. Individuals were then classified as short or long for each gene polymorphism on the basis of control median polymorphismlengths in order to further elucidate possible combined effects. No interaction associations between the three genes and transsexualism were identified. Conclusions This study provides evidence that male gender identity might be partly mediated through the androgen receptor. PMID:18962445

Full Text Available Anastomosis grouping, restriction fragment lengthpolymorphism (RFLP of the ITS regions including the 5.85 rDNA, and random amplified microsatellites (RAMS were used to characterize isolates of Rhizoctonia solani collected from Spain and Finland. There was a high similarity between the results obtained with the three techniques. RAMS markers revealed more genetic variation among isolates of R. solani than RFLP. The anastomosis group (AG–3 isolates were clearly separated from isolates belonging to other AGs by RAMS, RFLPs and anastomosis grouping. Almost all the isolates sampled from potato belonged to AG–3. No differences were observed between Spanish and Finnish AG–3 isolates.

Aims: To investigate the potential of amplified fragment lengthpolymorphism (AFLP) profiling for genotyping Arcobacter butzleri and to obtain further data on the genetic diversity of this organism. Methods and Results: Seventy-three isolates of Danish, British, Turkish, Swedish, Nigerian and Nor...

Wild populations of edible species are important source of genetic variability for cultivated lines that can undergo a drastic loss of diversity resulting from man's selection. The development of tools aimed at the clear-cut and safe identification and assessment of genetic variability of the wild and cultivated strains is thus a fundamental goal of molecular genetic research. In this study, we used two polymerase chain reaction (PCR)-based fingerprinting methods-amplified fragment lengthpolymorphism (AFLP) and restriction fragment lengthpolymorphism (RFLP) of laccase and manganese peroxidase genes-to assess genetic differences among strains and independently evolving lineages belonging to the Pleurotus eryngii complex. Both laccase RFLP and AFLP have been proved to distinguish unambiguously the three taxa studied: Pleurotus ferulae, P. eryngii, and P. eryngii var. nebrodensis. AFLP also showed enough sensitivity to detect polymorphisms among the strains, proving to be an efficient DNA fingerprinting tool in studies of strain assignment. The divergent RFLP laccase and manganese peroxidase patterns are also discussed in relation to the role played by these genes in the interaction between these fungi and their host plants.

Full Text Available A total of 17 isolates of Colletotrichum from alfalfa (Medicago sativa L. and red clover (Trifolium pratense L. plants with anthracnose symptoms were collected from 11 districts in Serbia during 2005-2010 and tested for variability in vegetative compatibility groups (VCGs and restriction fragment lengthpolymorphisms (RFLP. Nitrate nonutilising (nit mutants were isolated from each of investigated C. destructivum isolates by selecting chlorate-resistant sectors on medium with chlorate. The isolates were grouped in five VCGs while one isolate was self-incompatible. No relationship was found between VCGs and geographical origin of the isolates. Restriction Fragment LengthPolymorphism (RFLP analysis of a 900 bp intron of the glutamine synthetase (GS gene revealed a unique polymorphic profile of C. destructivum isolates, distinct from the profiles of other Colletotrichum species. An identical profile was produced for all C. destructivum isolates, regardless of their host and geographical origin. PCR-RFLP failed to detect some the Serbian C. destructivum isolates. [Projekat Ministarstva nauke Republike Srbije, br. TR31057

Amplified fragment lengthpolymorphism markers were used to assess genetic diversity in 10 male sterile wheat crop lines (hetero-cytoplasm with the same nucleus) in relation to a restorer wheat line. These male sterile lines were evaluated using 64 amplified fragment lengthpolymorphism primer combinations, and 13 primers produced polymorphic bands, generating a total 682 fragments. Of the 682 fragments, 113 were polymorphic. The polymorphic information content and marker index values demonstrated the utility of the primer combinations used in the present study. Unweighted pair group method with arithmetic mean and principal coordinate analysis of the genotypic data revealed clustering of accessions based on genetic relationships, and accessions were separated into 2 groups with their restorer line. Jaccard's similarity coefficient values suggested good variability among the male sterile lines, indicating their utility in breeding programs. The fallouts of analysis of molecular variance showed large within-group population variation, accounting for 77% of variation, while among-group comparison accounted for 23% of the total molecular variation, which was statistically significant. The molecular diversity observed in this study will be useful for selecting appropriate accessions for plant improvement and hybridization through molecular-breeding approaches and for developing suitable conservation strategies.

The purpose of this study was to evaluate fluorescent amplified fragment lengthpolymorphism (AFLP) analysis for the inter- and intraspecies differentiation of a collection of 96 strains of Listeria monocytogenes and 10 non- L. monocytogenes strains representing six other Listeria species...... of different origin. The AFLP technique was compared with three other molecular typing methods - ribotyping, random amplified polymorphic DNA analysis (RAPD), and pulsed-field gel electrophoresis (PFGE) - in terms of discriminatory ability. PCR-restriction fragment lengthpolymorphism was included....... Isolates with identical DNA profiles were distributed across the spectrum of origin. It was not possible to associate certain types with specific food sectors or clinical cases, which is indicative of the spread of L. monocytogenes clones across species. Overall, AFLP fingerprinting was suitable...

Our aim was to analyze the difference between methods for genotyping of Mycobacterium tuberculosis complex isolates. We collected genotyping results from Restriction Fragment LengthPolymorphism (RFLP) and Mycobacterial Interspersed Repetitive Units-Variable Numbers of Tandem Repeat (MIRU-VNTR) in a geographically limited area (Stockholm) during a period of three years. The number and proportion of isolates belonging to clusters was reduced by 45 and 35% respectively when combining the two methods compared with using RFLP or MIRU-VNTR only. The mean size of the clusters was smaller when combining methods and smaller with RFLP compared to MIRU-VNTR. In clusters with confirmed epidemiological links RFLP coincided slightly better than MIRU-VNTR but where there was a difference, the variation in MIRU-VNTR pattern was only in a single locus. In isolates with few IS6110 bands in RFLP, MIRU-VNTR differentiated the isolates more, dividing the RFLP clusters. Since MIRU-VNTR is faster and less labour-intensive it is the method of choice for routine genotyping. In most cases it will be sufficient for epidemiological purposes but true clustering might still be considered if there are epidemiological links and the MIRU-VNTR results differ in only one of its 24 loci.

Fourteen strains of Naegleria fowleri, two strains of N. gruberi, and one strain each of N. australiensis, N. jadini, N. lovaniensis, Acanthamoeba sp., A. castellanii, A. polyphaga, and A. comandoni isolated from patients, soil, or water were characterized by restriction fragment lengthpolymorphisms. Total cellular DNA (1 microgram) was digested with either HindIII, BglII, or EcoRI; separated on agarose gels; and stained with ethidium bromide. From 2 to 15 unusually prominent repetitive restriction fragment bands, totaling 15 to 50 kilobases in length and constituting probably more than 30% of the total DNA, were detected for all ameba strains. Each species displayed a characteristic pattern of repetitive restriction fragments. Digests of the four Acanthamoeba spp. displayed fewer, less intensely staining repetitive fragments than those of the Naegleria spp. All N. fowleri strains, whether isolated from the cerebrospinal fluid of patients from different parts of the world or from hot springs, had repetitive restriction fragment bands of similar total lengths (ca. 45 kilobases), and most repetitive bands displayed identical mobilities. However, polymorphic bands were useful in identifying particular isolates. Restriction fragment lengthpolymorphism analysis generally was consistent with taxonomy based on studies of infectivity, morphology, isoenzyme patterns, and antibody reactivity and suggests that this technique may help classify amebae isolated from clinical specimens or from the environment.

The prevention of fraudulent labeling of game meat constitutes an important part of food regulatory control and quality assurance systems. A polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP) analysis based on mitochondrial deoxyribonucleic acid (DNA) was developed for authentication of meats from chamois (Rupicapra rupicapra), pyrenean ibex (Capra pyrenaica), and mouflon (Ovis ammon). Amplification and restriction site analysis of a DNA fragment about 720 base pairs (bp) from the mitochondrial 12S rRNA gene of all analyzed species permitted the selection of Msel and Apol endonucleases for meat speciation. The 12S rRNA restriction profiles obtained allowed the unequivocal identification of chamois, pyrenean ibex, and mouflon/sheep and their differentiation from meats of domestic species such as cattle, goat, and swine. The highly variable mitochondrial D-loop gene was also targeted to attempt discrimination between mouflon and sheep meats. A D-loop region (700-1000 bp) was amplified and sequenced in all game and domestic species analyzed, and a primer set was designed for the selective amplification of a 370 bp DNA fragment from mouflon and sheep. PCR-RFLP analysis with the selected Maell enzyme generated a single electrophoretic profile characteristic for sheep, whereas 3 different fragment patterns were obtained for mouflon meats. Consequently, the PCR-RFLP technique developed can be routinely applied in inspection programs in order to verify the correct labeling of game species.

Full Text Available Abstract In order to effectively identify the vaccine and field strains of Canine distemper virus (CDV, a new differential diagnostic test has been developed based on reverse transcription-polymerase chain reaction (RT-PCR and restriction fragment lengthpolymorphism (RFLP. We selected an 829 bp fragment of the nucleoprotein (N gene of CDV. By RFLP analysis using BamHI, field isolates were distinguishable from the vaccine strains. Two fragments were obtained from the vaccine strains by RT-PCR-RFLP analysis while three were observed in the field strains. An 829 nucleotide region of the CDV N gene was analyzed in 19 CDV field strains isolated from minks, raccoon dogs and foxes in China between 2005 and 2007. The results suggest this method is precise, accurate and efficient. It was also determined that three different genotypes exist in CDV field strains in fur animal herds of the north of China, most of which belong to Asian type. Mutated field strains, JSY06-R1, JSY06-R2 and JDH07-F1 also exist in Northern China, but are most closely related to the standard virulent strain A75/17, designated in Arctic and America-2 genetype in the present study, respectively.

Feb 23, 2013 ... tion fragment lengthpolymorphism (RFLP) for amplified genomic DNA. The frequencies of the com- ... Meanwhile, the race selection should be paid more atten- tion since the pathogenesis of a disease might have different bases in different racial population groups. У 2013 Ain Shams University. Production ...

The genetic diversity of Mycoplasma capricolum subsp. capripneumoniae strains based on determination of amplified fragment lengthpolymorphisms (AFLP) is described. AFLP fingerprints of 38 strains derived from different countries in Africa and the Middle East consisted of over 100 bands in the size...... found by 16S rDNA analysis. The present data support previous observations regarding genetic homogeneity of M. capricolum subsp. capripneumoniae, and confirm the two evolutionary lines of descent found by analysis of 16S rRNA genes....

of different origin. The AFLP technique was compared with three other molecular typing methods - ribotyping, random amplified polymorphic DNA analysis (RAPD), and pulsed-field gel electrophoresis (PFGE) - in terms of discriminatory ability. PCR-restriction fragment lengthpolymorphism was included...... for virulence gene allele characterization. The 96 L. monocytogenes strains were divided into two major clusters by AFLP fingerprinting at a similarity level of 82% in concordance with the results of PFGE, RAPD, and ribotyping. One main cluster consisted of all of the 24 L. monocytogenes hly allele 1 strains...

The efficacy of polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP) analysis of mitochondrial 12S rRNA gene in identification of goat cashmere and sheep wool samples was evaluated. The specific fragments of the mitochondrial 12S rRNA gene, which were about 440 bp, were obtained using the PCR. Restriction enzyme digestion of the PCR products with endonucleases BspT I and Hinf I revealed species-specific RFLP patterns. Application of this technique on mixed samples could identify goat cashmere and sheep wool from each other within the proportion of 8:1. The technique, however, could detect only one species when the proportion of mixture was more than 9:1. The PCR-RFLP technique was demonstrated to possess potential value in precise identification of goat cashmere and sheep wool.

Full Text Available Genotypes of Mycobacterium tuberculosis complex (MTBC vary with the geographic origin of the patients and can affect tuberculosis (TB transmission. This study was aimed to further differentiate spoligotype-defined clusters of drug-resistant MTBC clinical isolates split in Beijing (n=190 versus non-Beijing isolates (n=84 from Sichuan region, the second high-burden province in China, by IS6110-restriction fragment lengthpolymorphism (RFLP and 24-locus MIRU-VNTRs. Among 274 spoligotyped isolates, the clustering ratio of Beijing family was 5.3% by 24-locus MIRU-VNTRs versus 2.1% by IS6110-RFLP, while none of the non-Beijing isolates were clustered by 24-locus MIRU-VNTRs versus 9.5% by IS6110-RFLP. Hence, neither the 24-locus MIRU-VNTR was sufficient enough to fully discriminate the Beijing family, nor the IS6110-RFLP for the non-Beijing isolates. A region adjusted scheme combining 12 highly discriminatory VNTR loci with IS6110-RFLP was a better alternative for typing Beijing strains in Sichuan than 24-locus MIRU-VNTRs alone. IS6110-RFLP was for the first time introduced to systematically genotype MTBC in Sichuan and we conclude that the region-adjusted scheme of 12 highly discriminative VNTRs might be a suitable alternative to 24-locus MIRU-VNTR scheme for non-Beijing strains, while the clusters of the Beijing isolates should be further subtyped using IS6110-RFLP for optimal discrimination.

Genotypes of Mycobacterium tuberculosis complex (MTBC) vary with the geographic origin of the patients and can affect tuberculosis (TB) transmission. This study was aimed to further differentiate spoligotype-defined clusters of drug-resistant MTBC clinical isolates split in Beijing (n = 190) versus non-Beijing isolates (n = 84) from Sichuan region, the second high-burden province in China, by IS6110-restriction fragment lengthpolymorphism (RFLP) and 24-locus MIRU-VNTRs. Among 274 spoligotyped isolates, the clustering ratio of Beijing family was 5.3% by 24-locus MIRU-VNTRs versus 2.1% by IS6110-RFLP, while none of the non-Beijing isolates were clustered by 24-locus MIRU-VNTRs versus 9.5% by IS6110-RFLP. Hence, neither the 24-locus MIRU-VNTR was sufficient enough to fully discriminate the Beijing family, nor the IS6110-RFLP for the non-Beijing isolates. A region adjusted scheme combining 12 highly discriminatory VNTR loci with IS6110-RFLP was a better alternative for typing Beijing strains in Sichuan than 24-locus MIRU-VNTRs alone. IS6110-RFLP was for the first time introduced to systematically genotype MTBC in Sichuan and we conclude that the region-adjusted scheme of 12 highly discriminative VNTRs might be a suitable alternative to 24-locus MIRU-VNTR scheme for non-Beijing strains, while the clusters of the Beijing isolates should be further subtyped using IS6110-RFLP for optimal discrimination. PMID:24724099

Full Text Available An alternative DNA detection system is based on the polymerase chain reaction (PCR amplification of a segment of the mitochondrial cytochrome b gene. Subsequent cleavage by a restriction enzymes gives rise to a specie-specific pattern on an agarose gel. We used five animal species (Mustela vison, Mustela putorius furo, Sus scrofa domesticus, Oryctolagus cuninculus, Anser anser. Length of PCR product was 359 bp and we used universal primers. Restriction fragment lengthpolymorphism was analyzed by using the restriction endonuclease AluI. Results of cleavage were visualized by using electrophoresis and UV transiluminator. Every animal specie has a unique combination of restriction fragments i.e. Mustela vison 81 bp, 109 bp and 169 bp, Mustela putorius furo 169 bp and 190 bp, Sus scrofa domesticus 115 bp and 244 bp, Oryctolagus cunninculus is not cleaved by AluI so it has whole 359 bp fragment on agarose gel, Anser anser 130 bp and 229 bp. The results suggest that the method of PCR - RFLP is rapid and simple method for identification of species. PCR – RFLP can reliably identify chosen species. Application of genetic methods is very useful for breeding of livestock and protection of biodiversity.

Full Text Available The objective of this work was to evaluate the genetic diversity, its organization and the genetic relationships within oil palm (Elaeis oleifera (Kunth Cortés, from America, and E. guineensis (Jacq., from Africa germplasm using Restriction Fragment LengthPolymorphism (RFLP and Amplified Fragment LengthPolymorphism (AFLP. In complement to a previous RFLP study on 241 E. oleifera accessions, 38 E. guineensis accessions were analyzed using the same 37 cDNA probes. These accessions covered a large part of the geographical distribution areas of these species in America and Africa. In addition, AFLP analysis was performed on a sub-set of 40 accessions of E. oleifera and 22 of E. guineensis using three pairs of enzyme/primer combinations. Data were subjected to Factorial Analysis of Correspondence (FAC and cluster analysis, with parameters of genetic diversity being also studied. Results appeared congruent between RFLP and AFLP. In the E. oleifera, AFLP confirmed the strong structure of genetic diversity revealed by RFLP, according to geographical origin of the studied material, with the identification of the same four distinct genetic groups: Brazil, French Guyana/Surinam, Peru, north of Colombia/Central America. Both markers revealed that genetic divergence between the two species is of the same magnitude as that among provenances of E. oleifera. This finding is in discrepancy with the supposed early tertiary separation of the two species.

ApoB is a large glycoprotein with an apparent molecular mass of 550 kDa on NaDodSO 4 /PAGE. Recently, apoB cDNA clones have been isolated from an expression library made with mRNA from a human hepatoma cell line. These clones, which were all 1.5-1.6 kilobases (kb) long and corresponded to the 3' end of apoB mRNA, were used to demonstrate that hepatic apoB mRNA is ≅ 22 kb long. In the current report, a probe derived from one of these cDNA clones, pB8, was used for in situ hybridization experiments to map the human gene for apoB, APOB, to the distal half of the short arm of chromosome 2. This probe was also used to analyze somatic cell hybrids and, in agreement with the in situ hybridization studies, concordancy was demonstrated with chromosome 2. In addition, two hybrids with chromosome 2 translocations that contain only the short arm reacted with the pB8 probe. A third hybrid with a complex rearrangement of chromosome 2, which deleted an interstitial region and the tip of the short arm of chromosome 2, did not react. These data indicate that APOB maps to either 2p21-p23 or 2p24-pter. In further studies, DNA from normal individuals, digested with the restriction endonuclease EcoRI and subjected to Southern blot analysis with the pB8 probe, revealed a two-allele restriction fragment lengthpolymorphism (RFLP). The mapping studies provide the means for understanding the relationship of the APOB locus to others in the human genome, whereas the demonstration of an APOB RFLP increases their ability to assess the role of this locus in determining plasma lipoprotein levels

Sep 6, 2010 ... We used the polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP) technique to screen for DNA polymorphisms of the leptin gene in 255 cows of Iranian Holstein. Amplified region is located in exon 3 of leptin gene. The genomic bovine leptin sequences, which consist of three ...

A group of 66 patients with preeclampsia and 72 control subjects were analyzed for the Arg16Gly and Gln27Glu polymorphisms of the ADRB2 by polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP). Comparisons of the ADRB2 genotypes or alleles between different groups were performed ...

Two Indica rice varieties, Bianpizhan and Xiangzhan, and their semi-dwarf mutants induced by high energy argon ion radiation, Ar-10, and Xiang-Ar-1, were examined with restriction fragment lengthpolymorphism (RFLP) analysis by using 97 rice single copy genomic clones mapped on 12 chromosomes of molecular genetic map, combined with 5 restriction enzymes. Among the markers screened, 9 detected polymorphism were between Bianpizhen and Ar-10, and 11 detected polymorphism were between Xiangzhan and Xiang-Ar-1. Moreover, two or more restriction enzymes could generate RFLP patterns when screened with a given marker for several polymorphic markers. Based on the polymorphic allelic loci, the mutation frequencies were estimated as 5.15% and 6.39% for Ar-10 and Xiang-Ar-1 respectively. These results suggested that the nature of mutation on the DNA level was probably large genetic changes rather than point mutation. Genetic analysis and gene tagging of semi-dwarf mutation in one of the mutant line, Ar-10, indicated that this mutation was controlled by a major recessive gene, which was preliminary located on chromosome 4

Two Indica rice varieties, Bianpizhan and Xiangzhan, and their semi dwarf mutants induced by high energy argon ion radiation, Ar 10, and Xiang Ar 1, were examined with restriction fragment lengthpolymorphism(RFLP)analysis by using 97 rice single copy genomic clones mapped on 12 chromosomes of molecular genetic map, combined with 5 restriction enzymes.Among the markers screened, 9 detected polymorphism were between Bianpizhan and Ar 10, and 11 detected polymorphism were between Xiangzhan and Xiang Ar 1.Moreover, two or more restriction enzymes could generate RFLP patterns when screened with a given marker for several polymorphic markers. Based on the polymorphic allelic loci, the mutation frequencies were estimated as 5 15% and 6 39% for Ar 10 and Xiang Ar 1 respectively.These results suggested that the nature of mutation on the DNA level was probably large genetic changes rather than point mutation.Genetic analysis and gene tagging of semi dwarf mutation in one of the mutant line, Ar 10, indicated that this mutation was controlled by a major recessive gene, which was preliminary located on chromosome 4. (author)

An amplified fragment lengthpolymorphism (AFLP) method, developed to genotype Yersinia enterocolitica, has been used to investigate 70 representative strains isolated from humans, pigs, sheep, and cattle in the United Kingdom. AFLP primarily distinguished Y enterocolitica strains according to th...

By Southern blot analysis, a restriction fragment lengthpolymorphism in the 3' flanking region of the rabbit beta 1-globin gene was detected. Two alleles, characterized by 9.7- and 12.4-kb BamHI fragments and by 15.3- and 18.0-kb HindIII fragments, have been detected in a small population of White New Zealand rabbits. The long allele is the most frequent (about 70%). The simultaneous changes in the restriction patterns of the two endonucleases and the constant distance between BamHI and HindIII sites in short and long fragments suggest the possibility that the two alleles arise from a rearrangement phenomenon involving a DNA segment 2.7 kb long. In addition, the presence of the two alleles in individuals genetically unrelated to the White New Zealand breed suggests that this polymorphism is widespread.

Cannabis sativa L. (Cannabaceae) is one of the earliest known cultivated plants and is important in the global economy today as a licit and an illicit crop. Molecular markers distinguishing licit and illicit cultivars have forensic utility, but no direct comparison of hemp and marijuana amplified fragment lengthpolymorphism (AFLP) has been made to date. Genetic variation was surveyed in three populations of fiber hemp and a potent cultivar of marijuana using AFLP markers. Ten primer pairs yielded 1206 bands, of which 88% were polymorphic. Eighteen bands represented fixed differences between all fiber populations and the drug cultivar. These markers have practical utility for (1) establishing conspiracy in the cultivation and distribution of marijuana, (2) identifying geographic sources of seized drugs, and (3) discriminating illegal, potent marijuana cultivars from hemp where the cultivation of industrial hemp is permitted.

The functional role of the exon 3 growth hormone receptor (d3GHR) polymorphism in human and its distributions in different populations is not clearly understood. The presence of full length growth hormone (flGHR) is the most important in metabolic risk factors. The aim of this study was to define the frequency distribution of d3GHR/full-length GHR in an Iranian population. The presence of the d3GHR polymorphism in healthy volunteers blood DNA (n=80, male=30 and female=50) was assessed by PCR using specific primers. The 935-bp and 592-bp fragments indicate the presence of the flGHR and the exon3 deletion of GHR, respectively. The distribution of the GHR genotypes in this study were 31.4% (n=24) for fl/flGHR, 49.7 % (n=41) for fl/d3GHR, and 19.0 % (n=15) for d3/d3GHR. Frequencies of fl allele and d3 allele were 55.4% and 44.4% within whole population, respectively. There was no difference in allels frequencies of GHR in male (fl=0.583, d3=0.417) and female (fl=0.540, d3=0.460) when compared with whole population. The results showed that the frequency of d3/d3GHR isoform was significantly lower than that of the fl/flGHR and d3/flGHR. The frequencies of GHR polymorphisms were likely consistent with previous reports. Our finding is also consistant with Mexican population. The advantage of existence of the d3/d3 rather than fl/flGHR polymorphisms in individuals and in correlation with diseases opens new insights for GH and insilin-like-growth factor-1 (IGF-I) axis.

A method for high-resolution genomic fingerprinting of the enteric pathogens Campylobacter jejuni and Campylobacter coli, based on the determination of amplified fragment lengthpolymorphism, is described. The potential of this method for molecular epidemiological studies of these species...... to available epidemiological data. We conclude that this amplified fragment lengthpolymorphism fingerprinting method may be a highly effective tool for molecular epidemiological studies of Campylobacter spp....

The pattern of genetic differentiation of the endemic alpine salamander, Salamandra atra, has been studied using amplified fragment lengthpolymorphism (AFLP) from 11 populations throughout the range of the two currently recognized subspecies, atra and aurorae. Five different primer combinations produced 706 bands and were analyzed by constructing a phylogenetic tree using NJ and principal component analysis. Significant genetic variation was revealed by AFLP between and within populations but, our results show a lack of genetic structure. AFLP markers seems to be unsuitable to investigate complex and recent diversification.

Genomic diversity among strains of Mycoplasma hyosynoviae isolated in Denmark was assessed by using amplified fragment lengthpolymorphism (AFLP) analysis. Ninety-six strains, obtained from different specimens and geographical locations during 30 years and the type strain of M. hyosynoviae S16(T......) were concurrently examined for variance in BglII-MfeI and EcoRI-Csp6I-A AFLP markers. A total of 56 different genomic fingerprints having an overall similarity between 77 and 96% were detected. No correlation between AFLP variability and period of isolation or anatomical site of isolation could...

There are approximately 20 known species of the genus Cryptosporidium, and among these, 8 infect immunocompetent or immunocompromised humans. C. hominis and C. parvum most commonly infect humans. Differentiating between them is important for evaluating potential sources of infection. We report here the development of a simple and accurate real-time PCR-based restriction fragment lengthpolymorphism (RFLP) method to distinguish between C. parvum and C. hominis. Using the CP2 gene as the target, we found that both Cryptosporidium species yielded 224 bp products. In the subsequent RFLP method using TaqI, 2 bands (99 and 125 bp) specific to C. hominis were detected. Using this method, we detected C. hominis infection in 1 of 21 patients with diarrhea, suggesting that this method could facilitate the detection of C. hominis infections.

The use of PCR-RFLP analysis as a useful microbial identification tool has been evaluated for years. This approach was verified effective worldwide, where differential DNA bands and sequence markers distinctive to specific microbes or microbial groups have been identified. In our study, PCR-RFLP technique has been adopted in the identification of our indigenous N 2 -fixing isolates obtained from several local environments. RFLP was carried out with suitable restriction enzymes and the patterns were documented. Representatives of the different patterns were selected and analysed with the 16S ribosomal DNA sequencing method. The results demonstrated correlation between the differential RFLP patterns and the 16S rDNA identities. (Author)

Full Text Available With advances in molecular genetics have been possible to predict the genetic value of the animal, in particular its potential to transmit desired characters to their offspring, including characters difficult to evaluate or with low heritability, as is the case of the meat tenderization. It is known that Bos taurus indicus features differences in meat tenderization, being assigned this variability to their lowest proteolysis post-mortem, as result of high activity of calpastatin. This inhibitor decreases the activity of calpain, which are the enzymes responsible for the degradation of muscle fibers during the maturation of the meat. Moreover, there were previously observed differences in the frequencies of allele A of calpain among European breeds (Hereford, Aberdeen Angus and Holstein and Bos taurus indicus (Gir, Guzerá and Nelore. This variability has been related to tenderness of meat, as cattle with Bos taurus taurus origin have more tender meat than Bos taurus indicus, showing small values of shear force. One explanation is that the Capn2A product could confer greater proteolytic activity than the encoded by the allele Capn2B. If allele A is associated with tender meat, it will be possible the early identification of the animals that have the potential to produce meat with qualities that attend the needs of the consumer market, in order to add economic value to the final product of the animal production chain. For this reason, biochemical and genetic studies related to calpain and calpastatin systems have been considered promising for the clarification of the physiological changes that occur in muscle structure during the period post-mortem, whose results have contributed to the improvement of meat quality. The objectives of this study were to investigate the RFLP in calpain (Capn2 gene and its relation with meat tenderization in 252 crossbred (Bos taurus taurus x Bos taurus indicus. The analyses were carried through by PCR-RFLP technique

Fusarium wilt, caused by Fusarium oxysporum Schlecht f. sp. melonis Snyder & Hans, is a worldwide soil-borne disease of melon (Cucumis melo L.). Resistance to races 0 and 1 of Fusarium wilt is conditioned by the dominant gene Fom-2. To facilitate marker-assisted backcrossing with selection for Fusarium wilt resistance, we developed cleaved amplified polymorphic sequences (CAPS) and restriction fragment lengthpolymorphisms (RFLP) markers by converting RAPD markers E07 (a 1.25-kb band) and G17 (a 1.05-kb band), respectively. The RAPD-PCR polymorphic fragments from the susceptible line 'Vedrantais' were cloned and sequenced in order to construct primers that would amplify only the target fragment. The derived primers, E07SCAR-1/E07SCAR-2 from E07 and G17SCAR-1/G17SCAR-2 from G17, yielded a single 1.25-kb fragment (designated SCE07) and a 1.05-kb fragment (designated SCG17) (the same as RAPD markers E07 and G17), respectively, from both resistant and susceptible melon lines, thus demonstrating locus-specific associated primers. Potential CAPS markers were first revealed by comparing sequence data between fragments amplified from resistant (PI 161375) and susceptible ('Vedrantais') lines and were then confirmed by electrophoresis of restriction endonuclease digestion products. Twelve restriction endonucleases were evaluated for their potential use as CAPS markers within the SCE07 fragment. Three (BclI, MspI, and BssSI) yielded ideal CAPS markers and were subsequently subjected to extensive testing using an additional 88 diverse melon cultigens, 93 and 119 F(2) individuals from crosses of 'Vedrantais' x PI 161375 and 'Ananas Yokneam'×MR-1 respectively, and 17 families from a backcross BC(1)S(1) population derived from the breeding line 'MD8654' as a resistance source. BclI- and MspI-CAPS are susceptible-linked markers, whereas the BssSI-CAPS is a resistant-linked marker. The CAPS markers that resulted from double digestion by BclI and BssSI are co-dominant. Results

Sugar beet plants regenerated from UV-treated calluses were examined by restriction fragment lengthpolymorphism (RFLP) analysis to determine the extent of somaclonal variation occurring at the DNA level. In total, 50 random sugar beet DNA sequences were used to screen 42 somaclones for genetic alterations. Three polymorphisms were detected among the 7 644 alleles analysed. From these data a mutation frequency of 0.03 ± 0.02% per allele was estimated. This frequency is in agreement with similar studies of somaclonal DNA variation using molecular markers and lies in the upper range of the spontaneous gene mutation frequencies found in plants. The two probegenotype combinations showing independent polymorphisms, were further analysed using the restriction enzymes Bam HI, Eco RI, Eco RV and Hind III. Both polymorphisms are likely to result from structural rearrangements rather than from point mutations. Differences in methylation among 10 of the investigated somaclones were tested for by comparing Hpa II and Msp I generated RFLP patterns. The somaclones showed extensive methylation, but no differences in their degree of methylation. Cytological analysis revealed 34 diploid, 8 tetraploid, but no aneuploid plants. (author)

A DNA fingerprinting method known as ALIS-FLP (amplified ligation selected fragment-lengthpolymorphism) has been developed for selective and specific amplification of restriction fragments from TspRI restriction endonuclease digested genomic DNA. The method is similar to AFLP, but differs......RI genomic DNA fragment, and a short, degenerated, oligonucleotide covering the remaining TspRI cohesive ends. Other cohesive ends are covered by a short degenerated oligonucleotide lacking the primer site. The ligation mixture is used as a template for amplification using a single primer corresponding...... for differentiation of the organisms without previous knowledge of their DNA sequence. The usefulness of the method is confirmed by genotyping of 70 previously characterized clinical E. coli isolates. The grouping obtained was identical to the results of REA-PFGE. Versatility of the method is highlighted, i.e. its...

Understanding how chromosomal alterations contribute to acquired and inherited human disease requires the ability to manage the enormous physical and informational complexity of the deoxyribonucleic acid (DNA) packaged within. Important concepts and techniques involved in the analysis of DNA include restriction enzymes, Southern blotting, and restriction fragment lengthpolymorphism/linkage analysis. These techniques have been essential in the understanding and diagnosis of several syndromes associated with the head and neck. The purpose of this article is to introduce DNA structure, describe some techniques fundamental to DNA analysis, and provide a brief overview of the clinical applications of this technology with respect to dentinogenesis imperfecta and oral field cancerization. Copyright 2001 American Association of Oral and Maxillofacial Surgeons.

The association between atherosclerosis and gut microbiota has been attracting increased attention. We previously demonstrated a possible link between gut microbiota and coronary artery disease. Our aim of this study was to clarify the gut microbiota profiles in coronary artery disease patients using data mining analysis of terminal restriction fragment lengthpolymorphism (T-RFLP). This study included 39 coronary artery disease (CAD) patients and 30 age- and sex- matched no-CAD controls (Ctrls) with coronary risk factors. Bacterial DNA was extracted from their fecal samples and analyzed by T-RFLP and data mining analysis using the classification and regression algorithm. Five additional CAD patients were newly recruited to confirm the reliability of this analysis. Data mining analysis could divide the composition of gut microbiota into 2 characteristic nodes. The CAD group was classified into 4 CAD pattern nodes (35/39 = 90 %), while the Ctrl group was classified into 3 Ctrl pattern nodes (28/30 = 93 %). Five additional CAD samples were applied to the same dividing model, which could validate the accuracy to predict the risk of CAD by data mining analysis. We could demonstrate that operational taxonomic unit 853 (OTU853), OTU657, and OTU990 were determined important both by the data mining method and by the usual statistical comparison. We classified the gut microbiota profiles in coronary artery disease patients using data mining analysis of T-RFLP data and demonstrated the possibility that gut microbiota is a diagnostic marker of suffering from CAD.

Steep vertical gradients of oxidants (O2 and NO3−) in Puget Sound and Washington continental margin sediments indicate that aerobic respiration and denitrification occur within the top few millimeters to centimeters. To systematically explore the underlying communities of denitrifiers, Bacteria, and Archaea along redox gradients at distant geographic locations, nitrite reductase (nirS) genes and bacterial and archaeal 16S rRNA genes (rDNAs) were PCR amplified and analyzed by terminal restriction fragment lengthpolymorphism (T-RFLP) analysis. The suitablility of T-RFLP analysis for investigating communities of nirS-containing denitrifiers was established by the correspondence of dominant terminal restriction fragments (T-RFs) of nirS to computer-simulated T-RFs of nirS clones. These clones belonged to clusters II, III, and IV from the same cores and were analyzed in a previous study (G. Braker, J. Zhou, L. Wu, A. H. Devol, and J. M. Tiedje, Appl. Environ. Microbiol. 66:2096–2104, 2000). T-RFLP analysis of nirS and bacterial rDNA revealed a high level of functional and phylogenetic diversity, whereas the level of diversity of Archaea was lower. A comparison of T-RFLPs based on the presence or absence of T-RFs and correspondence analysis based on the frequencies and heights of T-RFs allowed us to group sediment samples according to the sampling location and thus clearly distinguish Puget Sound and the Washington margin populations. However, changes in community structure within sediment core sections during the transition from aerobic to anaerobic conditions were minor. Thus, within the top layers of marine sediments, redox gradients seem to result from the differential metabolic activities of populations of similar communities, probably through mixing by marine invertebrates rather than from the development of distinct communities. PMID:11282647

Sources of Mycobacterium bovis contamination remain unclear for many cases of animal and human disease. A major limitation is the lack of sufficiently informative or epidemiologically well evaluated molecular methods for typing. Here, we report an evaluation of a high-throughput method based on 29 mycobacterial interspersed repetitive unit-variable-number tandem-repeat (MIRU-VNTR) loci to genotype 127 M. bovis isolates from cattle from 77 different Belgian farms, representative of a nationwide collection obtained from 1995 to 2003. MIRU-VNTR stability was demonstrated by analyzing a series of 74 isolates in total, obtained from different animals from a single farm or from different farms with an identified epidemiological link. The genotyping results and the genotypic diversity (h) were compared with those obtained by IS6110 restriction fragment lengthpolymorphism (RFLP) analysis and spoligotyping. Among 68 isolates with no known epidemiological link, MIRU-VNTR typing discriminated better than either RFLP analysis or spoligotyping, with isolates taken individually (32 versus 16 and 17 genotypes; h = 0.91 versus 0.73 and 0.85, respectively) or in combination (32 versus 28 genotypes; h = 0.91 versus 0.92). Maximal resolution was already achieved with a subset of 9 loci. The observed congruence of the genetic relationships based on IS6110 RFLP analysis, spoligotyping, and MIRU-VNTR markers is consistent with a clonal population structure of M. bovis. These results support MIRU-VNTR typing as a convenient and discriminatory technique for analysis of the population structure of M. bovis in much greater detail and for addressing some still unresolved issues in the epidemiology of the pathogen. PMID:16757584

The definitive genetic identification of Toxocara species is currently based on PCR/sequencing. The objectives of the present study were to design and conduct an in silico polymerase chain reaction-restriction fragment lengthpolymorphism method for identification of Toxocara species. In silico analyses using the DNASIS and NEBcutter softwares were performed with rDNA internal transcribed spacers, and mitochondrial cox1 and nad1 sequences obtained in our previous studies along with relevant sequences deposited in GenBank. Consequently, RFLP profiles were designed and all isolates of T. canis and T. cati collected from dogs and cats in different geographical areas of Iran were investigated with the RFLP method using some of the identified suitable enzymes. The findings of in silico analyses predicted that on the cox1 gene only the MboII enzyme is appropriate for PCR-RFLP to reliably distinguish the two species. No suitable enzyme for PCR-RFLP on the nad1 gene was identified that yields the same pattern for all isolates of a species. DNASIS software showed that there are 241 suitable restriction enzymes for the differentiation of T. canis from T. cati based on ITS sequences. RsaI, MvaI and SalI enzymes were selected to evaluate the reliability of the in silico PCR-RFLP. The sizes of restriction fragments obtained by PCR-RFLP of all samples consistently matched the expected RFLP patterns. The ITS sequences are usually conserved and the PCR-RFLP approach targeting the ITS sequence is recommended for the molecular differentiation of Toxocara species and can provide a reliable tool for identification purposes particularly at the larval and egg stages.

Phylogeographic and population genetic screens of chloroplast DNA (cpDNA) provide insights into seedbased gene flow in angiosperms, yet studies are frequently hampered by the low mutation rate of this genome. Detection methods for intraspecific variation can be either direct (DNA sequencing) or indirect (PCR-RFLP), although no single method incorporates the best...

The human cytomegalovirus (HCMV) a-sequence (a-seq) is located in the joining region between the long (L) and short (S) unique sequences of the virus (L-S junction), and this hypervariable junction has been used to differentiate HCMV strains. The purpose of this study was to investigate whether there are differences among strains of human cytomegalovirus which could be characterized by polymerase chain reaction (PCR) amplification of the a-seq of HCMV DNA and to compare a PCR method of strain differentiation with conventional restriction fragment lengthpolymorphism (RFLP) methodology by using HCMV junction probes. Laboratory strains of HCMV and viral isolates from individuals with HCMV infection were characterized by using both RFLPs and PCR. The PCR assay amplified regions in the major immediate-early gene (IE-1), the 64/65-kDa matrix phosphoprotein (pp65), and the a-seq of the L-S junction region. HCMV laboratory strains Towne, AD169, and Davis were distinguishable, in terms of size of the amplified product, when analyzed by PCR with primers specific for the a-seq but were indistinguishable by using PCR targeted to IE-1 and pp65 sequences. When this technique was applied to a characterization of isolates from individuals with HCMV infection, selected isolates could be readily distinguished. In addition, when the a-seq PCR product was analyzed with restriction enzyme digestion for the presence of specific sequences, these DNA differences were confirmed. PCR analysis across the variable a-seq of HCMV demonstrated differences among strains which were confirmed by RFLP in 38 of 40 isolates analyzed. The most informative restriction enzyme sites in the a-seq for distinguishing HCMV isolates were those of MnlI and BssHII. This indicates that the a-seq of HCMV is heterogeneous among wild strains, and PCR of the a-seq of HCMV is a practical way to characterize differences in strains of HCMV. Images PMID:1980680

Accurate identifying of the genotypes is considered one of the most important mechanisms used in the recording or the protection of plant varieties. The investigation was conducted at the experimental form belonging to the egyptian Atomic Energy Authority, Inshas. The aim was to evaluate grain quality characteristics and molecular genetic variation using Amplified Fragment LengthPolymorphism (AFLP) technique among six rice genotypes, Egyptian Jasmine aromatic rice cultivar and five aromatic rice mutants in (M3 mutagenic generation). Two mutation (Egy22 and Egy24) were selected from irradiated Sakha 102 population with 200 and 400Gy of gamma rays in the M2 generation, respectively, and three mutations ( Egy32, Egy33, and Egy34) were selected from irradiated Sakha 103 population with 200, 300, 400Gy of gamma rays in the M2 generation, respectively. The obtained results showed that the strong aroma was obtained for mutant Egy22 as compared with Egyptian Jasmine rice cultivar (moderate aroma). Seven primer combinations were used through six rice genotypes on the molecular level using AFLP marker. The size of AFLP Fragments Were Ranged from 51- 494bp. The total number of amplified bands was 997 band among them 919 polymorphic bans representing 92.2%. The highest similarity index (89%) was observed between Egyptian Jasmine and Egy32 followed by (82%) observed between Egyptian Jasmine and Egy34. On the other hand, the lowest similarity index was (48%) between Egyptian Jasmine and Egy24. In six rice genotypes, Egy24 produced the highest number of the AFLP makers giving 49 unique markers (23 positive and 26 negative), then Egy22 showed 23 unique markers (27 positive and 6 negative) while Egy33 was characterized by 17 unique markers (12 positive and 5 negative). At last Egyptian Jasmine was discriminated by the lowest number of markets, 10 (6 positive and 4 negative). The study further confirmed that AFLP technique was able to differentiate rice genotypes by a higher number

A high-density genetic map based on restriction fragment lengthpolymorphisms (RFLPs) is being constructed for loblolly pine (Pinus taeda L.). Consequently, a large number of DNA probes from loblolly pine are potentially available for use in other species. We have used some of these DNA probes to detect RFLPs in 12 conifers and an angiosperm....

Sea cucumbers are traditional marine food and Chinese medicine in Asia. The rapid expansion of sea cucumber market has resulted in various problems, such as commercial fraud and mislabeling. Conventionally, sea cucumber species could be distinguished by their morphological and anatomical characteristics; however, their identification becomes difficult when they are processed. The aim of this study was to develop a new convenient method of identifying and distinguishing sea cucumber species. Polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP) analysis of mitochondrial cytochrome oxidase I gene ( COI) was used to identifing five sea cucumber species ( Apostichopus japonicus, Cucumaria frondosa, Thelenota ananas, Parastichopus californicus and Actinopyga lecanora). A 692 bp fragment of COI was searched for BamHI, KpnI, PstI, XbaI and Eco31I restriction sites with DNAMAN 6.0, which were then used to PCR-RFLP analysis. These five sea cucumber species can be discriminated from mixed sea cucumbers. The developed PCR-RFLP assay will facilitate the identification of sea cucumbers, making their source tracing and quality controlling feasible.

There is evidence of antigenic diversity and of differences in virulence in Dermatophilus congolensis. For the understanding of the epidemiology of dermatophilosis it is important to distinguish between strains of the organism. Twenty field isolates from cattle in Chad and Cameroon, and an American reference strain, have been examined on restriction fragment lengthpolymorphisms. After restriction enzyme digestion of DNA by BamHI and Southern blotting, a rDNA probe consisting of plasmid pMC5 carrying a 4.8 kb insert of Mycoplasma capricolum DNA coding for the 5S, 23S and part of 16S rRNA allowed to distinguish 6 ribotypes of D. congolensis, based on their hybridized rDNA patterns. Particular ribotypes may be distributed over a wide geographical area. On the other hand, strains belonging to at least 5 different ribotypes may be found in one herd; this may partly explain the lack of success in immunization against dermatophilosis in the field.

A fast and reliable assay for the identification of dermatophyte fungi and nondermatophyte fungi (NDF) in onychomycosis is essential, since NDF are especially difficult to cure using standard treatment. Diagnosis is usually based on both direct microscopic examination of nail scrapings and macroscopic and microscopic identification of the infectious fungus in culture assays. In the last decade, PCR assays have been developed for the direct detection of fungi in nail samples. In this study, we describe a PCR-terminal restriction fragment lengthpolymorphism (TRFLP) assay to directly and routinely identify the infecting fungi in nails. Fungal DNA was easily extracted using a commercial kit after dissolving nail fragments in an Na(2)S solution. Trichophyton spp., as well as 12 NDF, could be unambiguously identified by the specific restriction fragment size of 5'-end-labeled amplified 28S DNA. This assay enables the distinction of different fungal infectious agents and their identification in mixed infections. Infectious agents could be identified in 74% (162/219) of cases in which the culture results were negative. The PCR-TRFLP assay described here is simple and reliable. Furthermore, it has the possibility to be automated and thus routinely applied to the rapid diagnosis of a large number of clinical specimens in dermatology laboratories.

Avian cholera, an infectious disease caused by the bacterium Pasteurella multocida, kills thousands of North American wild waterfowl annually. Pasteurella multocida serotype 1 isolates cultured during a laboratory challenge study of Mallards (Anas platyrhynchos) and collected from wild birds and environmental samples during avian cholera outbreaks were characterized using amplified fragment lengthpolymorphism (AFLP) analysis, a whole-genome DNA fingerprinting technique. Comparison of the AFLP profiles of 53 isolates from the laboratory challenge demonstrated that P. multocida underwent genetic changes during a 3-mo period. Analysis of 120 P. multocida serotype 1 isolates collected from wild birds and environmental samples revealed that isolates were distinguishable from one another based on regional and temporal genetic characteristics. Thus, AFLP analysis had the ability to distinguish P. multocida isolates of the same serotype by detecting spatiotemporal genetic changes and provides a tool to advance the study of avian cholera epidemiology. Further application of AFLP technology to the examination of wild bird avian cholera outbreaks may facilitate more effective management of this disease by providing the potential to investigate correlations between virulence and P. multocida genotypes, to identify affiliations between bird species and bacterial genotypes, and to elucidate the role of specific bird species in disease transmission. ?? Wildlife Disease Association 2008.

Full Text Available The aim of this study was to determine the genetic diversity of Giardia duodenalis present in a human population living in a northern Ecuadorian rain forest. All Giardia positive samples (based on an ELISA assay were analysed using a semi-nested polymerase chain reaction-restriction fragment lengthpolymorphism assay that targets the glutamate dehydrogenase (gdh gene; those amplified were subsequently genotyped using NlaIV and RsaI enzymes. The gdh gene was successfully amplified in 74 of 154 ELISA positive samples; 69 of the 74 samples were subsequently genotyped. Of these 69 samples, 42 (61% were classified as assemblage B (26 as BIII and 16 as BIV, 22 (32% as assemblage A (3 as AI and 19 as AII and five (7% as mixed AII and BIII types. In this study site we observe similar diversity in genotypes to other regions in Latin America, though in contrast to some previous studies, we found similar levels of diarrheal symptoms in those individuals infected with assemblage B compared with those infected with assemblage A.

To develop a novel polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP) protocol using a new genomic marker sequence and a novel set of restriction enzymes in order to detect and discriminate 2 Echinococcus species, E. granulosus and E. multilocularis, found in formalin-fixed paraffin-embedded (FFPE) human tissues. DNA was isolated from 11 FFPE human tissue samples positive for cystic echinococcosis or alveolar echinococcosis. A mitochondrial genomic marker region was amplified and sequenced using a novel primer pair and a new PCR-RFLP protocol was developed for the detection and discrimination of E. granulosus and E. multilocularis using a set of restriction enzymes including AccI, MboI, MboII, and TsoI. The selected marker region was amplified using DNA isolated from FFPE human tissue samples positive for cystic echinococcosis or alveolar echinococcosis and the discrimination of E. granulosus and E. multilocularis was accomplished by use of the novel PCR-RFLP method. In this PCR-RFLP protocol, use of any single restriction enzyme is enough for the discrimination of E. granulosus and E. multilocularis. The PCR-RFLP protocol can be potentially used for the discrimination of 5 other Echinococcus species: E. oligarthus, E. shiquicus, E. ortleppi, E. canadensis, and E. vogeli.

SNP (single nucleotide polymorphism) genotyping is the determination of genetic variations of SNPs between members of a species. In many laboratories, PCR-RFLP (polymerase chain reaction-restriction fragment lengthpolymorphism) is a usually used biotechnology for SNP genotyping, especially in small-scale basic research studies of complex genetic diseases. PCR-RFLP requires an available restriction enzyme at least for identify a target SNP and an effective primer pair conforms numerous constraints. However, the lots of restriction enzymes, tedious sequence and complicated constraints make the mining of available restriction enzymes and the design of effective primer pairs become a major challenge. In the study, we propose a novel and available CI (Computation Intelligence)-based method called TLBO (teaching-learning-based optimization) and introduce the elite strategy to design effective primer pairs. Three common melting temperature computations are available in the method. REHUNT (Restriction Enzymes HUNTing) is first combined with the method to mine available restriction enzymes. Robust in silico simulations for the GA (genetic algorithm), the PSO (particle swarm optimization), and the method for natural PCR-RFLP primer design in the SLC6A4 gene with two hundred and eighty-eight SNPs had been performed and compared. These methods had been implemented in JAVA and they are freely available at https://sites.google.com/site/yhcheng1981/tlbonpd-elite for users of academic and non-commercial interests.

Full Text Available Background: Clostridium tetani or Nicolaier’s bacillus is an obligatory anaerobic, Gram-positive, movable with terminal or sub terminal spore. The chromosome of C. tetani contains 2,799,250 bp with a G+C content of 28.6%. The aim of this study was identification and genomic fingerprinting of the vaccine strain of C. tetani.Materials and Methods: The vaccine strain of C. tetani was provided by Razi Vaccine and Serum Research Institute. The seeds were inoculated into Columbia blood agar and grown for 72 h and transferred to the thioglycolate broth medium for further 36 h culturing. The cultures were incubated at 35ºC in anaerobic conditions. DNA extraction with phenol/ chloroform method was performed. After extraction, the consistency of DNA was assayed. Next, the vaccine strain was digested using pvuII enzyme and incubated at 37ºC for overnight. The digested DNA was gel-electrophoresed by 1% agarose for a short time. Then, the gel was studied with Gel Doc system and transferred to Hybond N+membrane using standard DNA blotting techniques.Results: The vaccine strain of C. tetani genome was fingerprinted by RFLP technique. Our preliminary results showed no divergence exists in the vaccine strain used for the production tetanus toxoid during the periods of 1990-2011.Conclusion: Observation suggests that there is lack of significant changes in RFLP genomic fingerprinting profile of the vaccine strain. Therefore, this strain did not lose its efficiency in tetanus vaccine production. RFLP analysis is worthwhile in investigating the nature of the vaccine strain C. tetani.

In MeOH viewed as one of the improvement method for recovery of the petroleum with hope, the development of discrimination technique of press fit candidate microorganism and oil reservoir resident microorganism which exists in the test object oil reservoir was tried in order to monitor the survival situation of the microorganism which inserted in the oil reservoir under pressure. 16S rRNA amplified by the PCR using the universal primer The microorganism that it cut off the gene at restriction enzyme HhaI,MspI, AluI and inhabits oil reservoir water and oil reservoir rock in the object oil reservoir by ( necessarily TaqI ) and restriction fragment lengthpolymorphic analysis was classified. As the result, the effectiveness of the this PCR-RFLP method was indicated the microorganism which showed RFLP pattern which is identical with the press fit candidate microorganism in the oil reservoir resident microorganism for the discrimination of the press fit candidate microorganism without existing. And, it was indicated that the this PCR-RFLP method was effective for the investigation of oil reservoir resident microbial community which can positively utilize source of nutrition inserted to oil reservoir with the press fit candidate microorganism under pressure, and it was possible to grasp oil reservoir resident microorganism to be especially considered in MEOR. (translated by NEDO)

Full Text Available In order to compare their relative efficiencies as markers and to find the most suitable marker for maize diversity studies we evaluated 18 inbred tropical maize lines using a number of different loci as markers. The loci used were: 774 amplified fragment lengthpolymorphisms (AFLPs; 262 random amplified polymorphic DNAs (RAPDs; 185 restriction fragment lengthpolymorphisms (RFLPs; and 68 simple sequence repeats (SSR. For estimating genetic distance the AFLP and RFLP markers gave the most correlated results, with a correlation coefficient of r = 0.87. Bootstrap analysis were used to evaluate the number of loci for the markers and the coefficients of variation (CV revealed a skewed distribution. The dominant markers (AFLP and RAPD had small CV values indicating a skewed distribution while the codominant markers gave high CV values. The use of maximum values of genetic distance CVs within each sample size was efficient in determining the number of loci needed to obtain a maximum CV of 10%. The number of RFLP and AFLP loci used was enough to give CV values of below 5%, while the SSRs and RAPD loci gave higher CV values. Except for the RAPD markers, all the markers correlated genetic distance with single cross performance and heterosis which showed that they could be useful in predicting single cross performance and heterosis in intrapopulation crosses for broad-based populations. Our results indicate that AFLP seemed to be the best-suited molecular assay for fingerprinting and assessing genetic relationships among tropical maize inbred lines with high accuracy.

Many single nucleotide polymorphisms (SNPs) for complex genetic diseases are genotyped by polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP) in small-scale basic research studies. It is an essential work to design feasible PCR-RFLP primer pair and find out available restriction enzymes to recognize the target SNP for PCR experiments. However, many SNPs are incapable of performing PCR-RFLP makes SNP genotyping become unpractical. A genetic algorithm (GA) had been proposed for designing mutagenic primer and get available restriction enzymes, but it gives an unrefined solution in mutagenic primers. In order to improve the mutagenic primer design, we propose TLBOMPD (TLBO-based Mutagenic Primer Design) a novel computational intelligence-based method that uses the notion of "teaching and learning" to search for more feasible mutagenic primers and provide the latest available restriction enzymes. The original Wallace's formula for the calculation of melting temperature is maintained, and more accurate calculation formulas of GC-based melting temperature and thermodynamic melting temperature are introduced into the proposed method. Mutagenic matrix is also reserved to increase the efficiency of judging a hypothetical mutagenic primer if involve available restriction enzymes for recognizing the target SNP. Furthermore, the core of SNP-RFLPing version 2 is used to enhance the mining work for restriction enzymes based on the latest REBASE. Twenty-five SNPs with mismatch PCR-RFLP screened from 288 SNPs in human SLC6A4 gene are used to appraise the TLBOMPD. Also, the computational results are compared with those of the GAMPD. In the future, the usage of the mutagenic primers in the wet lab needs to been validated carefully to increase the reliability of the method. The TLBOMPD is implemented in JAVA and it is freely available at http://tlbompd.googlecode.com/.

The advent of complete genetic linkage maps consisting of codominant DNA markers [typically restriction fragment lengthpolymorphisms (RFLPs)] has stimulated interest in the systematic genetic dissection of discrete Mendelian factors underlying quantitative traits in experimental organisms. We describe here a set of analytical methods that modify and extend the classical theory for mapping such quantitative trait loci (QTLs). These include: (i) a method of identifying promising crosses for QTL mapping by exploiting a classical formula of SEWALL WRIGHT; (ii) a method (interval mapping) for exploiting the full power of RFLP linkage maps by adapting the approach of LOD score analysis used in human genetics, to obtain accurate estimates of the genetic location and phenotypic effect of QTLs; and (iii) a method (selective genotyping) that allows a substantial reduction in the number of progeny that need to be scored with the DNA markers. In addition to the exposition of the methods, explicit graphs are provided that allow experimental geneticists to estimate, in any particular case, the number of progeny required to map QTLs underlying a quantitative trait. PMID:2563713

The three main aims of the study were the assessment of the genetic relationship between a deviating Erwinia amylovora strain isolated from Amelanchier sp. (Maloideae) grown in Canada and other strains from Maloideae and Rosoideae, the investigation of the variability of the PstI fragment of the pEA29 plasmid using restriction fragment lengthpolymorphism (RFLP) analysis and the determination of the number of short-sequence DNA repeats (SSR) by DNA sequence analysis in representative strains. Ninety-three strains obtained from 12 plant genera and different geographical locations were examined by repetitive-sequences PCR using Enterobacterial Repetitive Intergenic Consensus, BOX and Repetitive Extragenic Palindromic primer sets. Upon the unweighted pair group method with arithmetic mean analysis, a deviating strain from Amelanchier sp. was analysed using amplified ribosomal DNA restriction analysis (ARDRA) analysis and the sequencing of the 16S rDNA gene. This strain showed 99% similarity to other E. amylovora strains in the 16S gene and the same banding pattern with ARDRA. The RFLP analysis of pEA29 plasmid using MspI and Sau3A restriction enzymes showed a higher variability than that previously observed and no clear-cut grouping of the strains was possible. The number of SSR units reiterated two to 12 times. The strains obtained from pear orchards showing for the first time symptoms of fire blight had a low number of SSR units. The strains from Maloideae exhibit a wider genetic variability than previously thought. The RFLP analysis of a fragment of the pEA29 plasmid would not seem a reliable method for typing E. amylovora strains. A low number of SSR units was observed with first epidemics of fire blight. The current detection techniques are mainly based on the genetic similarities observed within the strains from the cultivated tree-fruit crops. For a more reliable detection of the fire blight pathogen also in wild and ornamentals Rosaceous plants the genetic

Molecular methods are powerful tools in characterizing and determining relationships between plants. The aim of this study was to study genetic divergence between 103 accessions of Mexican Opuntia. To accomplish this, polymerase chain reaction (PCR)-restriction fragment lengthpolymorphism analysis of three chloroplast intergenic spacers (atpB-rbcL, trnL-trnF, and psbA-trnH), one chloroplast gene (ycf1), two nuclear genes (ppc and PhyC), and one mitochondrial gene (cox3) was conducted. The amplified products from all the samples had very similar molecular sizes, and there were only very small differences between the undigested PCR amplicons for all regions, with the exception of ppc. We obtained 5850 bp from the seven regions, and 136 fragments were detected with eight enzymes, 37 of which (27.2%) were polymorphic. We found that 40% of the fragments from the chloroplast regions were polymorphic, 9.8% of the bands detected in the nuclear genes were polymorphic, and 20% of the bands in the mitochondrial locus were polymorphic. trnL-trnF and psbA-trnH were the most variable regions. The Nei and Li/Dice distance was very short, and ranged from 0 to 0.12; indeed, 77 of the 103 genotypes had the same genetic profile. All the xoconostle accessions (acidic fruits) were grouped together without being separated from three genotypes of prickly pear (sweet fruits). We assume that the genetic divergence between prickly pears and xoconostles is very low, and question the number of Opuntia species currently considered in Mexico.

Increasing use of Mycoplasma gallisepticum (MG) live vaccines has led to a need for a rapid test for differentiation of MG field strains from the live vaccine strains ts-11 and 6/85. We examined the differentiating potential of diagnostic polymerase chain reaction (PCR) primers targeted to the gene mgc2, encoding a cytadherence-related surface protein uniquely present in MG. The mgc2-PCR diagnostic primers are specific for MG in tests of all avian mycoplasmas or bacteria present in the chicken trachea and are sensitive enough to readily detect MG in tracheal swabs from field outbreaks. Differentiation of vaccine strain ts-11 was based on identification of restriction enzyme sites in the 300-base-pair (bp) mgc2-PCR amplicon present in ts-11 and missing in MG isolates from field outbreaks in Israel. Restriction sites for the enzymes HaeII and SfaN1 were identified in the amplified region in strain ts-11 and were not found in 28 field isolates of MG, comprising a representative cross section of all the MG isolates from the period 1997-2003. In practice, differential diagnosis of MG is achieved within 1 day of submission of tracheal swab samples by mgc2-PCR amplification and restriction of the amplicon with HaeII, giving a 270-bp fragment for ts-11 or no restriction for other MG strains tested. Application of the mgc2-PCR-restriction fragment lengthpolymorphism (mgc2-PCR-RFLP) assay enabled differential diagnosis of both components of a mixture of ts-11 and non-ts-11 DNA, detecting the field strain in the presence of a large excess of ts-11. The test was successfully applied in vivo for monitoring vaccinates in a ts-11 vaccine trial. In principle, the test may also be used to identify the 6/85 vaccine strain, which yields a 237-bp product, readily differentiated from the approximately 300-bp PCR product of all other strains tested. Further testing of field isolates will be necessary to determine the applicability of this test in the United States and other countries.

Phytophthora capsici Leonian, an oomycete pathogen, is a serious problem in pepper worldwide. Its resistance in pepper is controlled by quantitative trait loci (QTL). To detect QTL associated with P. capsici resistance, a molecular linkage map was constructed using 100 F(2) individuals from a cross between Capsicum annuum 'CM334' and C. annuum 'Chilsungcho'. This linkage map consisted of 202 restriction fragment lengthpolymorphisms (RFLPs), 6 WRKYs and 1 simple sequence repeat (SSR) covering 1482.3 cM, with an average interval marker distance of 7.09 cM. QTL mapping of Phytophthora root rot and damping-off resistance was performed in F(2:3) originated from a cross between resistant Mexican landrace C. annuum 'CM334' and susceptible Korean landrace C. annuum 'Chilsungcho' using composite interval mapping (CIM) analysis. Four QTL explained 66.3% of the total phenotypic variations for root rot resistance and three 44.9% for damping-off resistance. Of these QTL loci, two were located close to RFLP markers CDI25 on chromosome 5 (P5) and CT211A on P9. A bacterial artificial chromosome (BAC) library from C. annuum 'CM334' was screened with these two RFLP probes to obtain sequence information around the RFLP marker loci for development of PCR-based markers. CDI25 and CT211 probes identified seven and eight BAC clones, respectively. Nine positive BAC clones containing probe regions were sequenced and used for cytogenetic analysis. One single-nucleotide amplified polymorphism (SNAP) for the CDI25 locus, and two SSRs and cleaved amplified polymorphic sequence (CAPS) for CT211 were developed using sequences of the positive BAC clones. These markers will be valuable for rapid selection of genotypes and map-based cloning for resistance genes against P. capsici.

Terminal restriction fragment lengthpolymorphism (T-RFLP) analysis is a common DNA-fingerprinting technique used for comparisons of complex microbial communities. Although the technique is well established there is no consensus on how to treat T-RFLP data to achieve the highest possible accuracy and reproducibility. This study focused on two critical steps in the T-RFLP data treatment: the alignment of the terminal restriction fragments (T-RFs), which enables comparisons of samples, and the normalization of T-RF profiles, which adjusts for differences in signal strength, total fluorescence, between samples. Variations in the estimation of T-RF sizes were observed and these variations were found to affect the alignment of the T-RFs. A novel method was developed which improved the alignment by adjusting for systematic shifts in the T-RF size estimations between the T-RF profiles. Differences in total fluorescence were shown to be caused by differences in sample concentration and by the gel loading. Five normalization methods were evaluated and the total fluorescence normalization procedure based on peak height data was found to increase the similarity between replicate profiles the most. A high peak detection threshold, alignment correction, normalization and the use of consensus profiles instead of single profiles increased the similarity of replicate T-RF profiles, i.e. lead to an increased reproducibility. The impact of different treatment methods on the outcome of subsequent analyses of T-RFLP data was evaluated using a dataset from a longitudinal study of the bacterial community in an activated sludge wastewater treatment plant. Whether the alignment was corrected or not and if and how the T-RF profiles were normalized had a substantial impact on ordination analyses, assessments of bacterial dynamics and analyses of correlations with environmental parameters. A novel method for the evaluation and correction of the alignment of T-RF profiles was shown to reduce

subspecies enterica isolates. A total of 25 serotypes were investigated that had been isolated from humans or veterinary sources in Denmark between 1995 and 2001. All isolates were genotyped by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and amplified fragment length...... polymorphism (AFLP). When making genetic trees, all three methods resulted in similar clustering that often corresponded with serotype, although some serotypes displayed more diversity than others. Of the three techniques, MLST was the easiest to interpret and compare between laboratories. Unfortunately...

A simple and robust biochemistry laboratory experiment is described that uses restriction fragment lengthpolymorphism (RFLP) of polymerase chain reaction (PCR) products to verify the identity of a potentially valuable horse. During the first laboratory period, students purify DNA from equine samples and amplify two loci of mitochondrial DNA. During the second laboratory period, students digest PCR products with restriction enzymes and analyze the fragment sizes through agarose gel electrophoresis. An optional step of validating DNA extracts through realtime PCR can expand the experiment to three weeks. This experiment, which has an engaging and versatile scenario, provides students with exposure to key principles and techniques of molecular biology, bioinformatics, and evolution in a forensic context.

Full Text Available Studies on the distribution of sand flies are important for the control of leishmaniasis in endemic and neighboring areas. In the present study polymerase chain reaction (PCR–restriction fragment lengthpolymorphism (RFLP was used to identify the distribution of sand flies in Al-Madinah and Asir Regions of Saudi Arabia using PCR–RFLP of 18S ribosomal RNA gene. Based on the morphological characteristics, the sand flies were differentiated into seven species viz., Phlebotomus papatasi, Phlebotomus sergenti, Phlebotomus bergeroti, Sergentomyia clydei, Sergentomyia antennata, Sergentomyia fallax and Sergentomyia schwetzi. PCR–RFLP of 18S ribosomal RNA (rRNA genes with eight different restriction enzymes resulted in species-specific agarose gel electrophoresis banding patterns. Of the eight restriction enzymes used, not a single restriction enzyme by itself could separate species belonging to the same genera (like P. papatasi and P. sergenti by AseI as well as those belonging to different genera (like P. papatasi and S. clydei by AseI. We therefore conclude that the genetic diversity within sand fly species based on PCR–RFLP technique was nonspecific. Studies are in progress to study the viability of alternate techniques like low-stringency single specific primer polymerase chain reaction which can be used for molecular typing.

While androgen and androgen receptor (AR) activity have been strongly implicated in prostate cancer development and therapy, the influence of the CAG repeat, which is found within the first exon of the AR gene, on prostate carcinogenesis is still unclear. We investigated the differences in the length of the CAG repeat between prostate cancer patients and controls in the Chinese population as well as between TMPRSS2:ERG fusion positive and negative samples. A general association between prostate cancer and either longer or shorter AR CAG repeat length was not observed in the Chinese population. However, our data suggest that certain CAG repeat lengths may increase or decrease prostate cancer risk. Shorter CAG repeat length was also not shown to be associated with a higher induction rate of TMPRSS2 and ERG proximity, an essential step for TMPRSS2:ERG fusion formation. However, samples with a CAG repeat of 17 were found more frequently in the TMPRSS2:ERG fusion positive than negative prostate cancer cases and mediated a higher rate of androgen-induced TMPRSS2 and ERG co-localisation than AR with longer (24) and shorter (15) CAG repeats. This suggests that 17 CAG repeats may be associated with TMPRSS2:ERG fusion positive prostate cancer, but may have a preventive role for prostate cancer in the Chinese population, which has a low TMPRSS2:ERG fusion frequency. This study suggests that different mechanisms for the association of CAG repeat lengthpolymorphism and prostate cancer exist in different ethnic populations. PMID:25520876

Although aggregation of Aβ amyloid fibrils into plaques in the brain is a hallmark of Alzheimer's Disease (AD), the correlation between amyloid burden and severity of symptoms is weak. One possible reason is that amyloid fibrils are structurally polymorphic and different polymorphs may contribute differentially to disease. However, the occurrence and distribution of amyloid polymorphisms in human brain is poorly documented. Here we seek to fill this knowledge gap by using X-ray microdiffraction of histological sections of human tissue to map the abundance, orientation and structural heterogeneities of amyloid within individual plaques; among proximal plaques and in subjects with distinct clinical histories. A 5 µ x-ray beam was used to generate diffraction data with each pattern arising from a scattering volume of only ~ 450 µ3 , making possible collection of dozens to hundreds of diffraction patterns from a single amyloid plaque. X-ray scattering from these samples exhibited all the properties expected for scattering from amyloid. Amyloid distribution was mapped using the intensity of its signature 4.7 Å reflection which also provided information on the orientation of amyloid fibrils across plaques. Margins of plaques exhibited a greater degree of orientation than cores and orientation around blood vessels frequently appeared tangential. Variation in the structure of Aβ fibrils is reflected in the shape of the 4.7 Å peak which usually appears as a doublet. Variations in this peak correspond to differences between the structure of amyloid within cores of plaques and at their periphery. Examination of tissue from a mismatch case - an individual with high plaque burden but no overt signs of dementia at time of death - revealed a diversity of structure and spatial distribution of amyloid that is distinct from typical AD cases. We demonstrate the existence of structural polymorphisms among amyloid within and among plaques of a single individual and suggest

Fourteen standard bred horses were serotyped as homozygous for 1 of 6 Equine Leukocyte Antigen (ELA) specificities. DNA was purified from peripheral leukocytes and digested with Hind III or Pvu II. Southern blot hybridization analysis was carried out using a 32 P-labeled mouse cDNA probe (PH2IIa) specific for class I MHC genes. Both enzymes generated blots that contained a large number of bands (23 to 30) per horse. Significant polymorphism existed among most fragment sizes, while a dozen highly conserved band sizes suggested the presence of Qa/tla - like genes. Only 2 animals (both W6's) showed identical band patterns. Polymorphism was greatest between horses of different serotypes and was significantly decreased within serotypes. Unique bands were present on both blots for both W1's and W6's and may account for the serologic specificity seen in ELA W1 and W6 horses. This study is consistent with the findings in other higher vertebrates and implies that the MHC of the horse includes a highly polymorphic class I multigene family

Full Text Available Introduction During a diagnostic evaluation of canine visceral leishmaniasis (VL, two of seventeen dogs were found to be co-infected by Leishmania (Viannia braziliensis and Leishmania (Leishmania chagasi. Methods Specific polymerase chain reaction (PCR and restriction fragment lengthpolymorphism-PCR (RFLP-PCR assays were performed. Results PCR assays for Leishmania subgenus identification followed by RFLP-PCR analysis in biopsies from cutaneous lesions and the spleen confirmed the presence of Leishmania (Viannia braziliensis and Leishmania (Leishmania chagasi in those fragments. Conclusions This report reinforces the importance of using serological and molecular techniques in the epidemiological surveillance of canine populations in endemic areas in which both diseases are known to co-exist. In such cases, a reassessment of the control measures is required.

During a diagnostic evaluation of canine visceral leishmaniasis (VL), two of seventeen dogs were found to be co-infected by Leishmania (Viannia) braziliensis and Leishmania (Leishmania) chagasi. Specific polymerase chain reaction (PCR) and restriction fragment lengthpolymorphism-PCR (RFLP-PCR) assays were performed. PCR assays for Leishmania subgenus identification followed by RFLP-PCR analysis in biopsies from cutaneous lesions and the spleen confirmed the presence of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) chagasi in those fragments. This report reinforces the importance of using serological and molecular techniques in the epidemiological surveillance of canine populations in endemic areas in which both diseases are known to co-exist. In such cases, a reassessment of the control measures is required.

Phytoplasmas were detected in cactus (Opuntia species) plants exhibiting witches'-broom disease symptoms in Yunnan Province, south-western China. Comparative and phylogenetic analyses of 16S rRNA gene sequences indicated that an overwhelming majority of the cactus-infecting phytoplasmas under study belonged to the peanut witches'-broom phytoplasma group (16SrII). Genotyping through use of computer-simulated restriction fragment lengthpolymorphism (RFLP) analysis of 16S rRNA genes revealed a remarkable genetic diversity among these cactus-infecting phytoplasma strains. Based on calculated coefficients of RFLP pattern similarities, seven new 16SrII subgroups were recognized, bringing the total of described group 16SrII subgroups to 12 worldwide. Geographical areas differed from one another in the extent of genetic diversity among cactus-infecting phytoplasma strains. The findings have implications for relationships between ecosystem distribution and the emergence of group 16SrII subgroup diversity.

Pemphigus vulgaris in Israeli Ashkenazi and non-Ashkenazi Jews and in Austrian non-Jewish patients is strongly associated with the DR4 and DRw6 alleles of the HLA-D region class II genes. Restriction fragment lengthpolymorphism analysis was undertaken with DQβ, DQα, and DRβ cDNA probes. Hybridization with the DQβ probe identifies Pvu II, BamHI, and EcoRV fragments that absolutely discriminate pemphigus vulgaris patients from healthy DR-, DQ-, and ethnic-matched controls. In contrast the DQα and DRβ probes failed to identify disease-specific restriction fragment lengthpolymorphism fragments. These studies indicate that DQw1 and DQw3 polymorphisms carried by pemphigus vulgaris patients may be directly involved in predisposition to the disease or may be tightly linked to the susceptibility gene itself. To our knowledge, this is the first example of an HLA restriction fragment lengthpolymorphism that is highly associated with susceptibility to autoimmune disease

The results of polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP) analysis, obtained after treatment of PCR-products with restriction endonuclease CfoI, could identify two members of the Anopheles maculipennis complex: An. maculipennis and An. artemievi. Treatment of amplification products with restriction endonuclease BsuI gave rise to fragment lengths of 192 and 218 bp, characteristic of An. artemievi, in the populations of the Talas (settlement of Kizil-Adyr, Kara-Bura District), Dzhelalabad (towns of Tashkumyr and Kara-Kul), and Osh (town of Gulcha, Alai District; village of Langar, Kara-Suisky District) Regions. After treatment of PCR-products with restriction endonuclease BstACI, fragment lengths of 292 and 150 bp, characteristic of An. messeae, were obtained for the mosquitoes of Issyk-Kul (town of Balykchi) and Naryn (settlement of Kochkorka, Kochkor District) Regions. To identify the molecular forms of An. superpictus, the investigators sequenced the amplification products obtained by PCR with 5.8S and 28S rRNA gene-specific primers. Analysis of the primary structure of the second internal transcribed spacer, by using the international databases, has indicated that molecular form X is prevalent in the study districts of Kyrgyzstan. The COI-COII region of the mitochondrial genome of the vector also underwent PCR-RFLP analysis. Three new haplotypes with restriction patterns of about 540, 420, 200, 150, 140 bp, about 540, 360, 280, 150, 140 bp, and about 580, 540, and 150, 140 bp have been identified along with the previously described haplotype X characterized by restriction products of 540, 420, 260, 150, and 140 bp in length.

Identification of Bactrocera carambolae Drew and Hancock, B. papayae Drew and Hancock, B. tau Walker, B. latifrons Hendel, B. cucurbitae Coquillett, B. umbrosa Fabricius and B. caudata Fabricius would pose a problem if only a body part or an immature stage were available. Analysis of polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP) of cytochrome oxidase I (COI) gene using primers COIR, COIF, UEA7 and UEA10 and restriction enzymes (MseI, RsaI and Alu1) was carried out. The banding profiles in the electrophoresis gel were analysed. The COI gene in six Bactrocera spp. was successfully amplified by COIR and COIF, as well as UEA7 and UEA10, while B. caudata was amplified successfully only by UEA primers. Using COI amplified PCR products and restriction enzymes, distinct banding profiles for B. tau, B. latifrons, B. cucurbitae and B. umbrosa were observed, but not for B. carambolae and B. papayae. However, using UEA7, UEA10 and RsaI, B. caudata could be identified, while B. carambolae and B. papayae might possibly be separated from one another. It was also shown that adult body parts or immature life stages of B. carambolae, B. papayae, B. latifrons and B. cucurbitae produced the same banding profiles as the adults. PCR-RFLP analyses are able to identify positively five Bactrocera species, while B. papayae and B. carambolae might possibly be separated from one another, even if immature life stages or adult body parts are used.

Leptospires are usually classified by methods based on DNA-DNA hybridization and the conventional cross-agglutination absorption test, which uses polyclonal antibodies against lipopolysaccharides. In this study, the amplification of the rpoB gene, which encodes the beta-subunit of RNA polymerase, was used as an alternative tool to identify Leptospira. DNA extracts from sixty-eight serovars were obtained, and the hypervariable region located between 1990 and 2500-bp in the rpoB gene was amplified by polymerase chain reaction (PCR). The 600-bp amplicons of the rpoB gene were digested with the restriction endonucleases TaqI, Tru1I, Sau3AI and MslI, and the restriction fragments were separated by 6% polyacrylamide gel electrophoresis. Thirty-five fragment patters were obtained from the combined data of restriction fragment lengthpolymorphism (PCR-RFLP) analysis and used to infer the phylogenetic relationships among the Leptospira species and serovars. The species assignments obtained were in full agreement with the established taxonomic classifications. Twenty-two serovars were effectively identified based on differences in their molecular profiles. However, the other 46 serovars remained clustered in groups that included more than one serovar of different species. This study demonstrates the value of RFLP analysis of PCR-amplified rpoB as an initial method for identifying Leptospira species and serovars.

Full Text Available Leptospires are usually classified by methods based on DNA-DNA hybridization and the conventional cross-agglutination absorption test, which uses polyclonal antibodies against lipopolysaccharides. In this study, the amplification of the rpoB gene, which encodes the beta-subunit of RNA polymerase, was used as an alternative tool to identify Leptospira. DNA extracts from sixty-eight serovars were obtained, and the hypervariable region located between 1990 and 2500-bp in the rpoB gene was amplified by polymerase chain reaction (PCR. The 600-bp amplicons of the rpoB gene were digested with the restriction endonucleases TaqI, Tru1I, Sau3AI and MslI, and the restriction fragments were separated by 6% polyacrylamide gel electrophoresis. Thirty-five fragment patters were obtained from the combined data of restriction fragment lengthpolymorphism (PCR-RFLP analysis and used to infer the phylogenetic relationships among the Leptospira species and serovars. The species assignments obtained were in full agreement with the established taxonomic classifications. Twenty-two serovars were effectively identified based on differences in their molecular profiles. However, the other 46 serovars remained clustered in groups that included more than one serovar of different species. This study demonstrates the value of RFLP analysis of PCR-amplified rpoB as an initial method for identifying Leptospira species and serovars.

In the present study, amplification refractory mutation system (ARMS) and polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP) methods were used for identification of recently described Serbia 1 (S1) and Peshter 1 (P1) mitochondrial haplotypes of Varroa destructor. Based on single nucleotide polymorphisms (SNPs) within cytochrome oxidase 1 (cox1) and cytochrome b (cytb) gene sequences, a total of 64 adult V. destructor females were analyzed from locations where the S1 and P1 haplotypes had been detected previously. Results of haplotype identification obtained by ARMS and PCR-RFLP methods were completely consistent with the sequencing data. Furthermore, in some analyzed samples the occurrence of site heteroplasmy at haplotype-defining sites was detected, as it was confirmed by double peaks in the sequence chromatograms. Neither mites with simultaneous nucleotide variability, nor those with combined SNP and heteroplasmy in cox1 and cytb were found. Given that this is the first occurrence of site heteroplasmy in V. destructor, the origin of this phenomenon and possible specific traits of heteroplasmic mites have yet to be determined.

Full Text Available Abstract Background Microbial forensics is important in tracking the source of a pathogen, whether the disease is a naturally occurring outbreak or part of a criminal investigation. Results A method and SPR Opt (SNP and PCR-RFLP Optimization software to perform a comprehensive, whole-genome analysis to forensically discriminate multiple sequences is presented. Tools for the optimization of forensic typing using Single Nucleotide Polymorphism (SNP and PCR-Restriction Fragment LengthPolymorphism (PCR-RFLP analyses across multiple isolate sequences of a species are described. The PCR-RFLP analysis includes prediction and selection of optimal primers and restriction enzymes to enable maximum isolate discrimination based on sequence information. SPR Opt calculates all SNP or PCR-RFLP variations present in the sequences, groups them into haplotypes according to their co-segregation across those sequences, and performs combinatoric analyses to determine which sets of haplotypes provide maximal discrimination among all the input sequences. Those set combinations requiring that membership in the fewest haplotypes be queried (i.e. the fewest assays be performed are found. These analyses highlight variable regions based on existing sequence data. These markers may be heterogeneous among unsequenced isolates as well, and thus may be useful for characterizing the relationships among unsequenced as well as sequenced isolates. The predictions are multi-locus. Analyses of mumps and SARS viruses are summarized. Phylogenetic trees created based on SNPs, PCR-RFLPs, and full genomes are compared for SARS virus, illustrating that purported phylogenies based only on SNP or PCR-RFLP variations do not match those based on multiple sequence alignment of the full genomes. Conclusion This is the first software to optimize the selection of forensic markers to maximize information gained from the fewest assays, accepting whole or partial genome sequence data as input. As

The structure of the human placental alkaline phosphatase gene from normal term placentae was studied by restriction enzyme digestion and Southern blot analysis using a cDNA probe to the gene for the placental enzyme. The DNA digests fall into three distinct patterns based on the presence and intensity of an extra 1.1-kilobase Pst I Band. The extra 1.1-kilobase band is present in 9 of 27 placenta samples, and in 1 of these samples the extra band is present at double intensity. No polymorphism was revealed by digestion with restriction enzymes EcoRI, Sma I, BamHI, or Sac I. The extra Pst I-digestion site may lie in a noncoding region of the gene because no correlation was observed between the restriction fragment lengthpolymorphism and the common placental alkaline phosphatase alleles identified by starch gel electrophoresis. In addition, because placental alkaline phosphatase is frequently re-expressed in neoplasms, the authors examined tissue from ovarian, testicular, and endometrial tumors and from BeWo choriocarcinoma cells in culture. The Pst I-DNA digestion patterns from these cells and tissues were identical to those seen in the normal ovary and term placentae. The consistent reproducible digestion patterns seen in DNA from normal and tumor tissue indicate that a major gene rearrangement is not the basis for the ectopic expression of placental alkaline phosphatase in neoplasia

Octopus minor (Sasaki, 1920) is an economically important cephalopod that is found in the northern coastal waters of China. In this study, we investigated genetic differentiation in fishery populations using amplified fragment lengthpolymorphisms (AFLPs). A total of 150 individuals were collected from five locations: Dalian (DL), Yan-tai (YT), Qingdao (QD), Lianyungang (LY), and Zhoushan (ZS), and 243 reproducible bands were amplified using five AFLP primer combinations. The percentage of polymorphic bands ranged from 53.33 to 76.08%. Nei's genetic identity ranged from 0.9139 to 0.9713, and the genetic distance ranged from 0.0291 to 0.0900. A phylogenetic tree was constructed using the unweighted pair group method with arithmetic mean, based on the genetic distance. The DL and YT populations originated from one clade, while the QD, LY, and ZS populations originated from another. The results indicate that the O. minor stock consisted of two genetic populations with an overall significantly analogous FST value (0.1088, P octopus fisheries, so that this marine resource can be conserved for its long-term utilization.

Full Text Available Marker assisted speed congenics technique is commonly used to facilitate backcrossing of mouse strains in nearly half the time it normally takes otherwise. Traditionally, the technique is performed by analyzing PCR amplified regions of simple sequence lengthpolymorphism (SSLP markers between the recipient and donor strains: offspring with the highest number of markers showing the recipient genome across all chromosomes is chosen for the next generation. Although there are well-defined panels of SSLP makers established between certain pairs of mice strains, they are incomplete for most strains. The availability of well-established marker sets for speed congenic screens would enable the scientific community to transfer mutations across strain backgrounds. In this study, we tested the suitability of over 400 SSLP marker sets among 10 mouse strains commonly used for generating genetically engineered models. The panel of markers presented here can readily identify the specified strains and will be quite useful in marker assisted speed congenic screens. Moreover, unlike newer single nucleotide polymorphism (SNP array methods which require sophisticated equipment, the SSLP markers panel described here only uses PCR and agarose gel electrophoresis of amplified products; therefore it can be performed in most research laboratories.

Marker assisted speed congenics technique is commonly used to facilitate backcrossing of mouse strains in nearly half the time it normally takes otherwise. Traditionally, the technique is performed by analyzing PCR amplified regions of simple sequence lengthpolymorphism (SSLP) markers between the recipient and donor strains: offspring with the highest number of markers showing the recipient genome across all chromosomes is chosen for the next generation. Although there are well-defined panels of SSLP makers established between certain pairs of mice strains, they are incomplete for most strains. The availability of well-established marker sets for speed congenic screens would enable the scientific community to transfer mutations across strain backgrounds. In this study, we tested the suitability of over 400 SSLP marker sets among 10 mouse strains commonly used for generating genetically engineered models. The panel of markers presented here can readily identify the specified strains and will be quite useful in marker assisted speed congenic screens. Moreover, unlike newer single nucleotide polymorphism (SNP) array methods which require sophisticated equipment, the SSLP markers panel described here only uses PCR and agarose gel electrophoresis of amplified products; therefore it can be performed in most research laboratories.

The authors have studied the genetic segregation of human T-cell receptor β-chain (TCRβ) genes on chromosome 7q in 40 CEPH (Centre d'Etude du Polymorphisme Humain) families by using restriction fragment lengthpolymorphisms (RFLPs). They constructed haplotypes from eight RFLPs by using variable- and constant-region cDNA probes, which detect polymorphisms that span more than 600 kilobases of the TCRβ gene complex. Analysis of allele distributions between TCRβ genes revealed significant linkage disequilibrium between only 6 of the 28 different pairs of RFLPs. This linkage disequilibrium strongly influences the most efficient order to proceed for typing of these RFLPs in order to achieve maximum genetic informativeness, which in this study revealed a 97.3% level of heterozygosity within the TCRβ gene complex. The results should provide new insight into recent reports of disease associations with the TCRβ gene complex and should assist in designing future experiments to detect or confirm the existence of disease-susceptibility loci in this region of the human genome

Full Text Available Abstract Background The National Bio Resource Project for the Rat in Japan (NBRP-Rat is focusing on collecting, preserving and distributing various rat strains, including spontaneous mutant, transgenic, congenic, and recombinant inbred (RI strains. To evaluate their value as models of human diseases, we are characterizing them using 109 phenotypic parameters, such as clinical measurements, internal anatomy, metabolic parameters, and behavioral tests, as part of the Rat Phenome Project. Here, we report on a set of 357 simple sequence lengthpolymorphism (SSLP markers and 122 rat strains, which were genotyped by the marker set. Results The SSLP markers were selected according to their distribution patterns throughout the whole rat genome with an average spacing of 7.59 Mb. The average number of informative markers between all possible pairs of strains was 259 (72.5% of 357 markers, showing their high degree of polymorphism. From the genetic profile of these rat inbred strains, we constructed a rat family tree to clarify their genetic background. Conclusion These highly informative SSLP markers as well as genetically and phenotypically defined rat strains are useful for designing experiments for quantitative trait loci (QTL analysis and to choose strategies for developing new genetic resources. The data and resources are freely available at the NBRP-Rat web site 1.

Full Text Available Although human T-lymphotropic virus type I (HTLV-I exhibits high genetic stability, as compared to other RNA viruses and particularly to human immunodeficiency virus (HIV, genotypic subtypes of this human retrovirus have been characterized in isolates from diverse geographical areas. These are currently believed not to be associated with different pathogenetic outcomes of infection. The present study aimed at characterizing genotypic subtypes of viral isolates from 70 HTLV-I-infected individuals from São Paulo, Brazil, including 42 asymptomatic carriers and 28 patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP, using restricted fragment lengthpolymorphism (RFLP analysis of long terminal repeat (LTR HTLV-I proviral DNA sequences. Peripheral blood mononuclear cell lysates were amplified by nested polymerase chain reaction (PCR and amplicons submitted to enzymatic digestion using a panel of endonucleases. Among HTLV-I asymptomatic carriers, viral cosmopolitan subtypes A, B, C and E were identified in 73.8%, 7.1%, 7.1% and 12% of tested samples, respectively, whereas among HAM/TSP patients, cosmopolitan A (89.3%, cosmopolitan C (7.1% and cosmopolitan E (3.6% subtypes were detected. HTLV-I subtypes were not statistically significant associated with patients' clinical status. We also conclude that RFLP analysis is a suitable tool for descriptive studies on the molecular epidemiology of HTLV-I infections in our environment.

Cultivation independent analyses of soil microbial community structures are frequently used to describe microbiological soil characteristics. This approach is based on direct extraction of total soil DNA followed by PCR amplification of selected marker genes and subsequent genetic fingerprint analyses. Semi-automated genetic fingerprinting techniques such as terminal restriction fragment lengthpolymorphism (T-RFLP) and ribosomal intergenic spacer analysis (RISA) yield high-resolution patterns of highly diverse soil microbial communities and hold great potential for use in routine soil quality monitoring, when rapid high throughput screening for differences or changes is more important than phylogenetic identification of organisms affected. Our objective was to perform profound statistical analysis to evaluate the cultivation independent approach and the consistency of results from T-RFLP and RISA. As a model system, we used two different heavy metal treated soils from an open top chamber experiment. Bacterial T-RFLP and RISA profiles of 16S rDNA were converted into numeric data matrices in order to allow for detailed statistical analyses with cluster analysis, Mantel test statistics, Monte Carlo permutation tests and ANOVA. Analyses revealed that soil DNA-contents were significantly correlated with soil microbial biomass in our system. T-RFLP and RISA yielded highly consistent and correlating results and both allowed to distinguish the four treatments with equal significance. While RISA represents a fast and general fingerprinting method of moderate cost and labor intensity, T-RFLP is technically more demanding but offers the advantage of phylogenetic identification of detected soil microorganisms. Therefore, selection of either of these methods should be based on the specific research question under investigation.

The aim of this study was to develop a standardized molecular assay that used limited resources and equipment for routine genotyping of isolates of the fish rhabdovirus, viral haemorrhagic septicaemia virus (VHSV). Computer generated restriction maps, based on 62 unique full-length (1524 nt......-gene by a set of three restriction enzymes was predicted to accurately enable the assignment of the VHSV isolates into the four major genotypes discovered to date. Further sub-typing of the isolates into the recently described sub-lineages of genotype I was possible by applying three additional enzymes...

Cotton is the world's leading textile fiber crop and is also grown as a bioenergy and food crop. Knowledge of the phylogeny of closely related species and the genome origin and evolution of polyploid species is significant for advanced genomics research and breeding. We have reconstructed the phylogeny of the cotton genus, Gossypium L., and deciphered the genome origin and evolution of its five polyploid species by restriction fragment analysis of repeated sequences. Nuclear DNA of 84 accessions representing 35 species and all eight genomes of the genus were analyzed. The phylogenetic tree of the genus was reconstructed using the parsimony method on 1033 polymorphic repeated sequence restriction fragments. The genome origin of its polyploids was determined by calculating the diploid-polyploid restriction fragment correspondence (RFC). The tree is consistent with the morphological classification, genome designation and geographic distribution of the species at subgenus, section and subsection levels. Gossypium lobatum (D7) was unambiguously shown to have the highest RFC with the D-subgenomes of all five polyploids of the genus, while the common ancestor of Gossypium herbaceum (A1) and Gossypium arboreum (A2) likely contributed to the A-subgenomes of the polyploids. These results provide a comprehensive phylogenetic tree of the cotton genus and new insights into the genome origin and evolution of its polyploid species. The results also further demonstrate a simple, rapid and inexpensive method suitable for phylogenetic analysis of closely related species, especially congeneric species, and the inference of genome origin of polyploids that constitute over 70 % of flowering plants.

To investigate the polymorphism distribution of vitamin D receptor (VDR) gene Fok I in Mongolian population of China. Polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP) method was used to analyze three genotypes FF, Ff and ff in the start codon of VDR gene (Fok I) in unrelated normal healthy Mongolian individuals of China. In the population, we obtained the allelic frequencies of 57% and 43% for (F) and (f) allele and the percentage of genotypes FF, Ff and ff to be 31%, 52%, and 17% respectively. The polymorphism frequency and distribution of this VDR gene Fok I in Mongolian population of China exhibit its own characteristics.

Full Text Available Eight isozyme systems were used in this study: acid phosphatase (ACP, alcohol dehydrogenase (ADH, esterase (EST, glutamate dehydrogenase (GDH, malate dehydrogenase (MDH, phosphoglucoisomerase (PGI, 6-phosphogluconate dehydrogenase (PGD, and phosphoglucomutase (PGM. The polymorphism of these enzyme systems was studied in 25 elite inbred lines. A total of 19 loci were identified, but only eight of them were polymorphic in the germplasm tested. The polymorphic index for the eight informative markers ranged from 0.08 to 0.57, with a mean value of 0.36. Five isozyme loci were mapped in F2:3 populations with existing RFLP data. Est-1, Gdh-2 and Pgi-2 were mapped to linkage groups 3, 14 and 9, respectively. As in previous reports, an ACP locus and a PGD locus were found to be linked, both located in linkage group 2 of the public sunflower map.

This study reports a rapid PCR-based technique using a one-enzyme RFLP for discrimination of yeasts isolated from bovine clinical and subclinical mastitis milk samples. We analyzed a total of 1,486 milk samples collected over 1 yr in south Sardinia and northern Italy, and 142 yeast strains were preliminarily grouped based on their cultural morphology and physiological characteristics. Assimilation tests were conducted using the identification kit API ID 32C and APILAB Plus software (bioMérieux, Marcy l'Etoile, France). For PCR-RFLP analysis, the 18S-ITS1-5.8S ribosomal(r)DNA region was amplified and then digested with HaeIII, and dendrogram analysis of RFLP fragments was carried out. Furthermore, within each of the groups identified by the API or PCR-RFLP methods, the identification of isolates was confirmed by sequencing of the D1/D2 region using an ABI Prism 310 automatic sequencer (Applied Biosystems, Foster City, CA). The combined phenotypic and molecular approach enabled the identification of 17 yeast species belonging to the genera Candida (47.9%), Cryptococcus (21.1%), Trichosporon (19.7%), Geotrichum (7.1%), and Rhodotorula (4.2%). All Candida species were correctly identified by the API test and their identification confirmed by sequencing. All strains identified with the API system as Geotrichum candidum, Cryptococcus uniguttulatus, and Rhodotorula glutinis also produced characteristic restriction patterns and were confirmed as Galactomyces geotrichum (a teleomorph of G. candidum), Filobasidium uniguttulatum (teleomorph of Crypt. uniguttulatus), and R. glutinis, respectively, by D1/D2 rDNA sequencing. With regard to the genus Trichosporon, preliminary identification by API was problematic, whereas the RFLP technique used in this study gave characteristic restriction profiles for each species. Moreover, sequencing of the D1/D2 region allowed not only successful identification of Trichosporon gracile where API could not, but also correct identification of

Full Text Available Four species of crabs of the genus Maja have been described along the European coast: M. brachydactyla, M. squinado, M. goltziana and M. crispata. The commercially important species M. brachydactyla and M. squinado achieve the largest body sizes and are the most similar in morphology, and are therefore easily confused. The four species of Maja were identified using a novel morphometric index and a polymerase chain reaction followed by restriction fragment lengthpolymorphism analysis (RFLP. The relationship between carapace length and the distance between the tips of antorbital spines was used to distinguish adults of M. brachydactyla and M. squinado. PCR-RFLP analysis of a partial sequence of the mitochondrial cytochrome oxidase type I (COI revealed that the four species of the genus Maja can be unambiguously discriminated using the combination of restriction endonucleases enzymes HpyCH4V and Ase I. The molecular identification may be particularly useful in larvae, juvenile and young crabs, when the morphological differences found in adults are not applicable.

chain reaction-restriction fragment lengthpolymorphism of the flagellin A gene (flaA-RFLP) to determine their genetic relationships. Ten distinct clusters were recognized by flaA-RFLP typing. The results showed that horizontal transmission was the major route of Campylobacter transmission in this study. In conclusion, the emergence of MDR and high resistance rates to several antimicrobials are major concerns identified in this study. The prudent use of these agents and active surveillance of resistance at the farm level are essential steps to reduce the public health risks identified in this work.

Deficiency of human fumarylacetoacetase (FAH) activity results in hereditary tyrosinemia type I. Using the restriction enzymes BglII, KpnI and StuI and a 1.3-kb cDNA probe for the FAH gene, we have found 6 restriction fragment lengthpolymorphisms (RFLPs). These RFLPs were utilised in 3 tyrosinemia

The power to detect departures from the theoretical proportion of new mutants in X-linked lethal disorders has been analyzed for several types of segregation analysis, including methods based on completely linked restriction fragment lengthpolymorphisms. It is shown that all methods require large

Members of the genus Colletotrichum include some of the most economically important fungal pathogens in the world. Accurate diagnosis is critical to devising disease management strategies. Two species, Colletotrichum gloeosporioides and C. truncatum, are responsible for anthracnose disease in papaya (Carica papaya L.) and bell pepper (Capsicum annuum L.) in Trinidad. The ITS1-5.8S-ITS2 region of 48 Colletotrichum isolates was sequenced, and the ITS PCR products were analyzed by PCR-RFLP analysis. Restriction site polymorphisms generated from 11 restriction enzymes enabled the identification of specific enzymes that were successful in distinguishing between C. gloeosporioides and C. truncatum isolates. Species-specific restriction fragment lengthpolymorphisms generated by the enzymes AluI, HaeIII, PvuII, RsaI, and Sau3A were used to consistently resolve C. gloeosporioides and C. truncatum isolates from papaya. AluI, ApaI, PvuII, RsaI, and SmaI reliably separated isolates of C. gloeosporioides and C. truncatum from bell pepper. PvuII, RsaI, and Sau3A were also capable of distinguishing among the C. gloeosporioides isolates from papaya based on the different restriction patterns that were obtained as a result of intra-specific variation in restriction enzyme recognition sites in the ITS1-5.8S-ITS2 rDNA region. Of all the isolates tested, C. gloeosporioides from papaya also had the highest number of PCR-RFLP haplotypes. Cluster analysis of sequence and PCR-RFLP data demonstrated that all C. gloeosporioides and C. truncatum isolates clustered separately into species-specific clades regardless of host species. Phylograms also revealed consistent topologies which suggested that the genetic distances for PCR-RFLP-generated data were comparable to that of ITS sequence data. ITS PCR-RFLP fingerprinting is a rapid and reliable method to identify and differentiate between Colletotrichum species.

Loss of heterozygosity (LOH) has been extensively studied on the short arm of chromosome 3, and functional proofs have been obtained defining a tumor-suppressor locus at 3p21-22. We examined 31 paraffin-embedded cervical cancer samples for LOH, using 5 PCR-primer pairs, located around 3p21. Allele loss was found in 19 out of the 27 informative samples (70%) while 13 out of 23 informative samples (56%) had LOH located at 3p21-22. More of the human papillomavirus (HPV)-positive samples had LOH compared to the HPV-negative samples, giving only a weak association between loss of allele and HPV integration. Modifications of the DNA in the formaldehyde-fixed samples were detected, and further studies will be required to clarify how such artifacts may affect restriction fragment lengthpolymorphism (RFLP) studies on fixed tissues.

Full Text Available Mymensingh is the most endemic district for kala-azar in Bangladesh. Phlebotomus argentipes remains the only known vector although a number of sand fly species are prevalent in this area. Genotyping of sand flies distributed in a VL endemic area was developed by a PCR and restriction-fragment-lengthpolymorphism (RFLP of 18S rRNA gene of sand fly species. Using the RFLP-PCR analysis with AfaI and HinfI restriction enzymes, P. argentipes, P. papatasi, and Sergentomyia species could be identified. Among 1,055 female sand flies successfully analyzed for the species identification individually, 64.4% flies was classified as Sergentomyia species, whereas 35.6% was identified as P. argentipes and no P. papatasi was found. Although infection of Leishmania within the sand flies was individually examined targeting leishmanial minicircle DNA, none of the 1,055 sand flies examined were positive for Leishmania infection. The RFLP-PCR could be useful tools for taxonomic identification and Leishmania infection monitoring in endemic areas of Bangladesh.

Full Text Available Restriction fragment lengthpolymorphism (RFLP analysis of a PCR-amplified fragment of the 16S rRNA gene was performed on reference strains belonging to 21 different enterococcal species and on 75 Enterococcus isolates recovered from poultry meat, pasteurised milk and fresh cheese. PCR amplification generated a 275 bp fragment, which was digested with three restriction endonucleases (DdeI, HaeIII, HinfI. The strains were divided into five groups (groups A-E on the basis of their restriction patterns. Five biochemical tests (arabinose, arginine, manitol, methyl-β-D-glucopyranoside and raffinose were then performed in addition to RFLP analysis to narrow the identification of enterococcal strains to the species level. PCR-RFLP, in conjunction with the selected biochemical tests, allowed the precise identification of the 21 species of Enterococcus included in the present study. This proposed method is relatively simple and rapid and can be useful as an adjunct tool for accurate identification of Enterococcus.

A reliable data set is a fundamental prerequisite for consistent results and conclusions in population genetic studies. However, marker scoring of genetic fingerprints such as amplified fragment lengthpolymorphisms (AFLPs) is a highly subjective procedure, inducing inconsistencies owing to personal or laboratory-specific criteria. We applied two alternative marker selection algorithms, the newly developed script scanAFLP and the recently published AFLPScore, to a large AFLP genome scan to test how population genetic parameters and error rates were affected. These results were confronted with replicated random selections of marker subsets. We show that the newly developed marker selection criteria reduced the mismatch error rate and had a notable influence on estimates of genetic diversity and differentiation. Both effects are likely to influence biological inference. For example, genetic diversity (HS) was 29% lower while genetic differentiation (FST) was 8% higher when applying scanAFLP compared with AFLPScore. Likewise, random selections of markers resulted in substantial deviations of population genetic parameters compared with the data sets including specific selection criteria. These randomly selected marker sets showed surprisingly low variance among replicates. We conclude that stringent marker selection and phenotype calling reduces noise in the data set while retaining patterns of population genetic structure.

Full Text Available Mycobacterium avium is an important pathogen among immunodeficient patients, especially patients with AIDS. The natural history of this disease is unclear. Several environmental sources have been implicated as the origin of this infection. Polyclonal infection with this species is observed, challenging the understanding of its pathogenesis and treatment. In the present study 45 M. avium strains were recovered from 39 patients admitted to a reference hospital between 1996 and 1998. Species identification was performed using a species-specific nucleic acid hybridization test (AccuProbe® from Gen-Probe®. Strains were genotyped using IS1245 restriction fragment lengthpolymorphism typing. Blood was the main source of the organism. In one patient with disseminated disease, M. avium could be recovered more than once from potentially sterile sites. Strains isolated from this patient had different genotypes, indicating that the infection was polyclonal. Four patient clones were characterized in this population, the largest clone being detected in eight patients. This finding points to a common-source transmission of the organism.

A total of 131 phlebotomine Algerian sandflies have been processed in the present study. They belong to the species Phlebotomus bergeroti, Phlebotomus alexandri, Phlebotomus sergenti, Phlebotomus chabaudi, Phlebotomus riouxi, Phlebotomus perniciosus, Phlebotomus longicuspis, Phlebotomus perfiliewi, Phlebotomus ariasi, Phlebotomus chadlii, Sergentomyia fallax, Sergentomyia minuta, Sergentomyia antennata, Sergentomyia schwetzi, Sergentomyia clydei, Sergentomyia christophersi and Grassomyia dreyfussi. They have been characterised by sequencing of a part of the cytochrome b (cyt b), t RNA serine and NADH1 on the one hand and of the cytochrome C oxidase I of the mitochondrial DNA (mtDNA) on the other hand. Our study highlights two sympatric populations within P. sergenti in the area of its type-locality and new haplotypes of P. perniciosus and P. longicuspis without recording the specimens called lcx previously found in North Africa. We tried to use a polymerase chain reaction-restriction fragment lengthpolymorphism method based on a combined double digestion of each marker. These method is not interesting to identify sandflies all over the Mediterranean Basin.

The stable fly, Stomoxys calcitrans (L.), is a cosmopolitan pest of livestock and humans. The pestiferous nature and painful bite cause stress to cattle and other animals. The stress and resulting avoidance behaviors manifest as reductions in weight gain or milk production in cattle; estimated annual economic loss in the United States exceeds US$2 billion. Understanding the population genetics of stable flies could provide information on their population dynamics, origins of outbreaks, and geographical patterns of insecticide resistance, resulting in a tactical advantage for developing management strategies. Previous studies, mostly on a local scale, reported a high level of gene flow between locations. Here, we report results wherein amplified fragment lengthpolymorphism was used to determine genetic diversity of stable fly samples consisting of 11-40 individuals from 12 locations representing the United States, Canada, and Panama. The Analysis of Molecular Variance showed that the majority of genetic diversity was within groups; very little was among groups. The F(ST) and G(ST) values were low ( 1.0). The tests of neutrality suggested population expansion, and no genetic differentiation was found between locations. These results show that stable flies have a high level of gene flow on a continental scale, with limited isolation owing to distance or geographical barriers.

Full Text Available A total of 131 phlebotomine Algerian sandflies have been processed in the present study. They belong to the species Phlebotomus bergeroti, Phlebotomus alexandri, Phlebotomus sergenti, Phlebotomus chabaudi, Phlebotomus riouxi, Phlebotomus perniciosus, Phlebotomus longicuspis, Phlebotomus perfiliewi, Phlebotomus ariasi, Phlebotomus chadlii, Sergentomyia fallax, Sergentomyia minuta, Sergentomyia antennata, Sergentomyia schwetzi, Sergentomyia clydei, Sergentomyia christophersi and Grassomyia dreyfussi. They have been characterised by sequencing of a part of the cytochrome b (cyt b, t RNA serine and NADH1 on the one hand and of the cytochrome C oxidase I of the mitochondrial DNA (mtDNA on the other hand. Our study highlights two sympatric populations within P. sergenti in the area of its type-locality and new haplotypes of P. perniciosus and P. longicuspis without recording the specimens called lcx previously found in North Africa. We tried to use a polymerase chain reaction-restriction fragment lengthpolymorphism method based on a combined double digestion of each marker. These method is not interesting to identify sandflies all over the Mediterranean Basin.

An amplified fragment lengthpolymorphism (AFLP) method, developed to genotype Yersinia enterocolitica, has been used to investigate 70 representative strains isolated from humans, pigs, sheep, and cattle in the United Kingdom. AFLP primarily distinguished Y. enterocolitica strains according to their biotype, with strains dividing into two distinct clusters: cluster A, comprising largely the putatively pathogenic biotypes (BT2 to -4), and cluster B, comprising the putatively nonpathogenic biotype 1A strains and a single BT1B isolate. Within these two clusters, subclusters formed largely on the basis of serotype. However, AFLP profiles also allowed differentiation of strains within these serotype-related subclusters, indicating the high discriminatory power of the technique for Y. enterocolitica. Investigation of the relationship between strain AFLP profile and host confirmed that pigs are, and provides further proof that sheep may be, potential sources of human infection with putatively pathogenic strains. However, the results suggest that some strains causing human disease do not come from veterinary sources identifiable at this time. The distribution of some BT1A isolates within cluster A raises questions about the relationship between virulence potential and biotype. PMID:16151073

A descriptive study was conducted to identify the different strains of Echinococcus granulosus occurring in livestock in Moroto district, Uganda. Echinococcus cysts from 104 domestic animals, including cattle, sheep, goats and camels, were taken and examined by microscopy, polymerase chain reaction with restriction fragment lengthpolymorphism and Sanger DNA sequencing. Echinococcus granulosus genotypes or strains were identified through use of Bioinformatics tools: BioEdit, BLAST and MEGA6. The major finding of this study was the existence of a limited number of E. granulosus genotypes from cattle, goats, sheep and camels. The most predominant genotype was G1 (96.05%), corresponding to the common sheep strain. To a limited extent (3.95%), the study revealed the existence of Echinococcus canadensis G6/7 in three (n = 3) of the E. granulosus-positive samples. No other strains of E. granulosus were identified. It was concluded that the common sheep strain of Echinococcus sensu stricto and G6/7 of E. canadensis were responsible for echinococcal disease in Moroto district, Uganda.

Bois noir phytoplasma (BNp), widespread in wine-producing areas of Europe and endemic in France and Italy, is classified in the 16SrXII-A subgroup, whose members are referred to as Stolbur phytoplasmas. The 16S rDNA gene of Stolbur phytoplasma shows low variability, and few non-ribosomal genes are available as markers to assess variation among isolates. We used the Stolbur-specific stol-1H10 gene, encoding a putative membrane-exposed protein, to investigate genetic diversity of French and Italian BNp isolates from plants and insects. Amplification of stol-1H10 from infected grapevines, weeds, and Hyalesthes obsoletus produced fragments of three sizes, and restriction fragment lengthpolymorphism analysis divided these amplicons further into 12 profiles (V1 to V12). French BNp isolates were more variable than Italian ones, and different profiles were present in infected grapevines from France and Italy. Isolate V3, most abundant among Italian affected grapes but present among French ones, was found in one Urtica dioica sample and in all H. obsoletus collected on this species. Four Italian-specific profiles were represented among infected Convolvulus arvensis, the most frequent of which (V12) was also detected in H. obsoletus collected on this species. Most of the variability in the stol-1H10 sequence was associated with type II on the tuf gene.

Full Text Available Angiostrongylus cantonensis, A. costaricensis, and A. vasorum are etiologic agents of human parasitic diseases. Their identification, at present, is only possible by examining the adult worm after a 40-day period following infection of vertebrate hosts with the third-stage larvae. In order to obtain a diagnostic tool to differentiate larvae and adult worm from the three referred species, polymerase chain reaction-restriction fragment lengthpolymorphism was carried out. The rDNA second internal transcribed spacer (ITS2 and mtDNA cytochrome oxidase I regions were amplified, followed by digestion of fragments with the restriction enzymes RsaI, HapII, AluI, HaeIII, DdeI and ClaI. The enzymes RsaI and ClaI exhibited the most discriminating profiles for the differentiation of the regions COI of mtDNA and ITS2 of rDNA respectively. The methodology using such regions proved to be efficient for the specific differentiation of the three species of Angiostrongylus under study.

Insulin-like growth factor binding protein-3 (IGFBP-3) gene is important for regulation of growth and development in mammals. The present investigation was carried out to study DNA polymorphism by PCR-RFLP of IGFBP-3 gene and its effect on fibre traits of Chinese Inner Mongolian cashmere goats. The fibre traits data investigated were cashmere fibre diameter, combed cashmere weight, cashmere fibre length and guard hair length. Four hundred and forty-four animals were used to detect polymorphis...

Pemphigus vulgaris in Israeli Ashkenazi and non-Ashkenazi Jews and in Austrian non-Jewish patients is strongly associated with the DR4 and DRw6 alleles of the HLA-D region class II genes. Restriction fragment lengthpolymorphism analysis was undertaken with DQ..beta.., DQ..cap alpha.., and DR..beta.. cDNA probes. Hybridization with the DQ..beta.. probe identifies Pvu II, BamHI, and EcoRV fragments that absolutely discriminate pemphigus vulgaris patients from healthy DR-, DQ-, and ethnic-matched controls. In contrast the DQ..cap alpha.. and DR..beta.. probes failed to identify disease-specific restriction fragment lengthpolymorphism fragments. These studies indicate that DQw1 and DQw3 polymorphisms carried by pemphigus vulgaris patients may be directly involved in predisposition to the disease or may be tightly linked to the susceptibility gene itself. To our knowledge, this is the first example of an HLA restriction fragment lengthpolymorphism that is highly associated with susceptibility to autoimmune disease.

Full Text Available Abstract Background Sesame is an important oil crop in tropical and subtropical areas. Despite its nutritional value and historic and cultural importance, the research on sesame has been scarce, particularly as far as its genetic diversity is concerned. The aims of the present study were to clarify genetic relationships among 32 sesame accessions from the Venezuelan Germplasm Collection, which represents genotypes from five diversity centres (India, Africa, China-Korea-Japan, Central Asia and Western Asia, and to determine the association between geographical origin and genetic diversity using amplified fragment lengthpolymorphism (AFLP. Results Large genetic variability was found within the germplasm collection. A total of 457 AFLP markers were recorded, 93 % of them being polymorphic. The Jaccard similarity coefficient ranged from 0.38 to 0.85 between pairs of accessions. The UPGMA dendrogram grouped 25 of 32 accessions in two robust clusters, but it has not revealed any association between genotype and geographical origin. Indian, African and Chinese-Korean-Japanese accessions were distributed throughout the dendrogram. A similar pattern was obtained using principal coordinates analysis. Genetic diversity studies considering five groups of accessions according to the geographic origin detected that only 20 % of the total diversity was due to diversity among groups using Nei's coefficient of population differentiation. Similarly, only 5% of the total diversity was attributed to differences among groups by the analysis of molecular variance (AMOVA. This small but significant difference was explained by the fact that the Central Asia group had a lower genetic variation than the other diversity centres studied. Conclusion We found that our sesame collection was genetically very variable and did not show an association between geographical origin and AFLP patterns. This result suggests that there was considerable gene flow among diversity centres

Background Sesame is an important oil crop in tropical and subtropical areas. Despite its nutritional value and historic and cultural importance, the research on sesame has been scarce, particularly as far as its genetic diversity is concerned. The aims of the present study were to clarify genetic relationships among 32 sesame accessions from the Venezuelan Germplasm Collection, which represents genotypes from five diversity centres (India, Africa, China-Korea-Japan, Central Asia and Western Asia), and to determine the association between geographical origin and genetic diversity using amplified fragment lengthpolymorphism (AFLP). Results Large genetic variability was found within the germplasm collection. A total of 457 AFLP markers were recorded, 93 % of them being polymorphic. The Jaccard similarity coefficient ranged from 0.38 to 0.85 between pairs of accessions. The UPGMA dendrogram grouped 25 of 32 accessions in two robust clusters, but it has not revealed any association between genotype and geographical origin. Indian, African and Chinese-Korean-Japanese accessions were distributed throughout the dendrogram. A similar pattern was obtained using principal coordinates analysis. Genetic diversity studies considering five groups of accessions according to the geographic origin detected that only 20 % of the total diversity was due to diversity among groups using Nei's coefficient of population differentiation. Similarly, only 5% of the total diversity was attributed to differences among groups by the analysis of molecular variance (AMOVA). This small but significant difference was explained by the fact that the Central Asia group had a lower genetic variation than the other diversity centres studied. Conclusion We found that our sesame collection was genetically very variable and did not show an association between geographical origin and AFLP patterns. This result suggests that there was considerable gene flow among diversity centres. Future germplasm

Full Text Available Toxoplasma gondii is a worldwide prevalent parasite, affecting a wide range of mammals and human beings. Little information is available about the distribution of genetic diversity of T. gondii infection in minks (Neovison vison. This study was conducted to estimate the prevalence and genetic characterization of T. gondii isolates from minks in China. A total of 418 minks brain tissue samples were collected from Jilin and Hebei provinces, northern China. Genomic DNA were extracted and assayed for T. gondii infection by semi-nested PCR of B1 gene. The positive DNA samples were typed at 10 genetic markers (SAG1, SAG2 (5'+3' SAG2, alter.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico using polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP technology. 36 (8.6% of 418 DNA samples were overall positive for T. gondii. Among them, 5 samples were genotyped at all loci, and 1 sample was genotyped for 9 loci. In total, five samples belong to ToxoDB PCR-RFLP genotype#9, one belong to ToxoDB genotye#3. To our knowledge, this is the first report of genetic characterization of T. gondii in minks in China. Meanwhile, these results revealed a distribution of T. gondii infection in minks in China. These data provided base-line information for controlling T. gondii infection in minks.

Toxoplasma gondii is a worldwide prevalent parasite, affecting a wide range of mammals and human beings. Little information is available about the distribution of genetic diversity of T. gondii infection in minks (Neovison vison). This study was conducted to estimate the prevalence and genetic characterization of T. gondii isolates from minks in China. A total of 418 minks brain tissue samples were collected from Jilin and Hebei provinces, northern China. Genomic DNA were extracted and assayed for T. gondii infection by semi-nested PCR of B1 gene. The positive DNA samples were typed at 10 genetic markers (SAG1, SAG2 (5'+3' SAG2, alter.SAG2), SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) using polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP) technology. 36 (8.6%) of 418 DNA samples were overall positive for T. gondii. Among them, 5 samples were genotyped at all loci, and 1 sample was genotyped for 9 loci. In total, five samples belong to ToxoDB PCR-RFLP genotype#9, one belong to ToxoDB genotye#3. To our knowledge, this is the first report of genetic characterization of T. gondii in minks in China. Meanwhile, these results revealed a distribution of T. gondii infection in minks in China. These data provided base-line information for controlling T. gondii infection in minks.

Authentication of meat assumes significance in view of religious, quality assurance, food safety, public health, conservation and legal concerns. Here, we describe a PCR-RFLP (Polymerase Chain Reaction- Restriction Fragment LengthPolymorphism) assay targeting mitochondrial cytochrome-b gene for the identification of meats of five most common food animals namely cattle, buffalo, goat, sheep and pig. A pair of forward and reverse primers (VPH-F & VPH-R) amplifying a conserved region (168-776 bp) of mitochondrial cytochrome-b (cytb) gene for targeted species was designed which yielded a 609 bp PCR amplicon. Further, restriction enzyme digestion of the amplicons with Alu1 and Taq1 restriction enzymes resulted in a distinctive digestion pattern that was able to discriminate each species. The repeatability of the PCR-RFLP assay was validated ten times with consistent results observed. The developed assay can be used in routine diagnostic laboratories to differentiate the meats of closely related domestic livestock species namely cattle from buffalo and sheep from goat.

Background: Somatic mutations in isocitrate dehydrogenase 1 (1DH1) gene occur frequently in primary brain tumors. Recently theses mutations were demonstrated in acute myeloid leukemia (AML). So far, assessment of these mutations relied on the DNA sequencing technique. Aim of the work: The aim of this study was to detect somatic mutations in IDH1 gene using mismatched primers suitable for endonuclease based detection, without the need for DNA sequencing, and to estimate its prognostic value, on patients with de novo AML. Methods: Residual DNA extracted from pretreatment bone marrow (BM) samples of 100 patients with de novo AML was used. The polymerase chain reaction-restriction fragment lengthpolymorphism method (PCR-RFLP) was adapted to IDHl gene, codon 132 mutations screening. Results: The frequency of IDH1 mutations was 13%. In the non-acute promyelocytic leukemia group (non-APL), IDH1 mutations were significantly associated with FLT3-ITD negative patients (p = 0.03). Patients with 1DH1 mutations did not achieve complete remission (CR). There was a trend for shorter overall survival (OS) in patients with IDH1 mutation compared to those with wild type (p = 0.08). Conclusion: IDH1 mutations are recurring genetic alterations in AML and they may have unfavorable impact on clinical outcome in adult AML. The PCR-RFLP method allows for a fast, inexpensive, and sensitive method for the detection of IDF11 mutations in AML.

Full Text Available Reticulitermes termites play key roles in dead wood decomposition and nutrient cycling in forests. They also damage man-made structures, resulting in considerable economic loss. In the eastern United States, five species (R. flavipes, R. virginicus, R. nelsonae, R. hageni and R. malletei have overlapping ranges and are difficult to distinguish morphologically. Here we present a molecular tool for species identification. It is based on polymerase chain reaction (PCR amplification of a section of the mitochondrial cytochrome oxidase subunit II gene, followed by a three-enzyme restriction fragment lengthpolymorphism (RFLP assay, with banding patterns resolved via agarose gel electrophoresis. The assay was designed using a large set of training data obtained from a public DNA sequence database, then evaluated using an independent test panel of Reticulitermes from the Southern Appalachian Mountains, for which species assignments were determined via phylogenetic comparison to reference sequences. After refining the interpretive framework, the PCR-RFLP assay was shown to provide accurate identification of four co-occurring species (the fifth species, R. hageni, was absent from the test panel, so accuracy cannot yet be extended to training data. The assay is cost- and time-efficient, and will help improve knowledge of Reticulitermes species distributions.

Full Text Available We developed a nested-PCR followed by restriction fragment lengthpolymorphism (RFLP for the detection of human cytomegalovirus (HCMV UL97 M460V/I, H520Q, C592Q,A594V, L595S/F and C603W mutations associated to ganciclovir (GCV resistance.The method uses five primer pairs and seven enzymes already published and newly combined.The detection limit of nPCR was assessed in a single serial dilution assay to be about 0.13 PFU/reaction. Expected restriction fragment patterns were obtained by nPCR-RFLP on either wild-type reference strains or strains and sequences of HCMV containing mutations. Then the nPCR-RFLP was used on 24 sera/plasma belonging to 22 transplant recipients (kidney, bone marrow, or kidney-pancreas: 13 subjects never treated with GCV (control group and 9 subjects treated with GCV oral profilaxis (study group. All codons detected from the control group (six in 8 cases and four in 1 case were identified as wild-type. All codons detected from the study group (six in 6 cases, three in 2 cases, and four in the second sample of 1 case whose first sample was negative by nPCR were wild-type except one, which showed a restriction pattern referring to M460V and/or M460I ATA-codified, definitively proved to be M460V by sequence analysis.This was the case of a renal transplant recipient at the end of profilaxis. In conclusion, the procedure seems to be quite sensitive and specific as well as able to detect mixed population of mutants or mutants and wild-type. It could represent a good tool in monitoring the emergence of HCMV mutants in renal transplant recipients treated with GCV.

Restriction length fragment polymorphism of the L-MYC gene was examined in DNAs from lung cancer tissues and normal tissues of 51 Japanese patients with lung cancer. In individual patients, no difference was seen between the restriction length fragments of the two alleles of L-MYC [6-kilobase (kb)] and 10-kb fragments in EcoRI digests in lung cancer tissues and normal tissues. But a striking correlation was found between the restriction length fragment polymorphism pattern of L-MYC and the extent of metastasis, particularly to the lymph nodes at the time of surgery: Patients with only the L band (10 kb) had few lymph node metastatic lesions, whereas patients with either the S band (6 kb) or the S and L bands almost always had lymph node metastatic lesion. A similar correlation was found between the presence of the S band and metastases to other organs. This correlation was particularly marked in cases of adenocarcinoma. These results indicate a clear genetic influence on metastases and a consequent poor prognosis for certain patients of lung cancer; L-MYC restriction length fragment polymorphism is thus shown to be a useful marker for predicting the metastatic potential of human lung cancer

The objective of this study was to identify restriction fragment lengthpolymorphism (RFLP) markers linked to Quantitative Trait Loci (QTL) that control aluminum (Al) tolerance in maize. The strategy used was bulked segregant analysis (BSA) and the genetic materials utilized were the F 2 , F 3 and F 4 populations derived from a cross between the Al-susceptible inbred line L53 and Al-tolerant inbred line L1327. The populations were evaluated in a nutrient solution containing a toxic concentration of Al (6 ppm) and relative seminal root length (RSRL) was used as a phenotypic measure of tolerance. Seedlings of the F 2 population with the highest and lowest RSRL values were transplanted to the field and subsequently selfed to obtain F 3 and F 4 families. The efficiency of the phenotypic index for selection was found to be greater when mean values were used instead of individual RSRL values. F 3 and F 4 families were then evaluated in nutrient solution to identify those that were not segregating. One hundred and thirteen probes, with an average interval of 30 cM, covering the 10 maize chromosomes were tested for their ability to discriminate the parental lines. Fifty four of these probes were polymorphic with 46 showing codominance. These probes were hybridized with DNA from two F 3 contrasting, bulks and three probes on chromosome 8 were found to be able distinguish the F 3 contrasting bulks on the basis of band position and intensity. DNA of families from the F 3 bulks hybridized with these probes showed the presence of heterozygous individuals. These three selected probes were also hybridized with DNA from F 2 individuals. Two of them showed a significant regression coefficient with the character. However, each of these probes explained only about 10% of the phenotypic variance observed in 70 F 2 individuals. One of the probes UMC 103 was hybridized with DNA from 168 F 4 families and the regression analysis of RFLP data showed a significant regression coefficient

Colletotrichum gloeosporioides sensu lato is one of the most common and widely distributed plant pathogens in the world. Understanding fungal biodiversity is hinged on accurate identification and delimitation at the inter- and intraspecific levels. Sequences of the ITS1-5.8S-ITS2 region (ITS), β-tubulin (TUB), actin (ACT), and glyceraldehyde-3-phosphate dehydrogenase (GPDH) genes of 30 C. gloeosporioides sensu lato isolates, collected from anthracnose infected papaya fruits grown in the main production areas in Trinidad, were analyzed by in silico PCR-RFLP analysis with the aim of identifying which gene region(s) had the highest level of intraspecific polymorphism. Restriction site polymorphisms generated from 13 restriction enzymes enabled the identification of specific enzymes that were successful at intraspecific discrimination of the C. gloeosporioides isolates. Genetic distance values were reflective of the level of polymorphisms obtained for the four different gene regions. In both cases (calculated genetic distance and percentage of polymorphic loci from RFLP profiles), ACT and ITS gene regions had the highest level of restriction site polymorphisms and genetic diversity, GPDH and TUB had the lowest. Cluster analysis based on PCR-RFLP genetic distance data revealed sub-specific placement of the isolates which appeared to be gene-dependent. The implications of these findings are discussed relative to biodiversity monitoring and the need for multilocus, polyphasic investigations which must take into account the possibility of exaggerated estimates of genetic diversity.

SDF-1 G801A polymorphism is reported to correlate with cancer susceptibility. However, the association between SDF-1 G801A polymorphism and non-small cell lung cancer (NSCLC) risk in Chinese populations remains unknown. A total of 408 NSCLC patients and 303 health controls included in this study. Restriction length fragment polymorphism (RFLP) analysis was used to assess the frequencies of SDF-1 G801A polymorphic variant. No significant association was found between SDF-1 G801A polymorphism and NSCLC risk (OR=1.268, 95% CI 0.811-2.583, P=0.361). Furthermore, SDF-1 G801A polymorphism was not correlated with histological type (P=0.697) and TNM stage (P=0.276). SDF-1 G801A polymorphism was not a risk factor for NSCLC in Chinese Han population.

The soft rot bacteria Erwinia carotovora and Erwinia chrysanthemi are important pathogens of potato and other crops. However, the taxonomy of these pathogens, particularly at subspecies level, is unclear. An investigation using amplified fragment lengthpolymorphism (AFLP) fingerprinting was undertaken to determine the taxonomic relationships within this group based on their genetic relatedness. Following cluster analysis on the similarity matrices derived from the AFLP gels, four clusters (c...

Terminal restriction fragment lengthpolymorphism analysis of reverse-transcribed 16S rRNA during periods of community flux was used as a tool to delineate the roles of the members of a 2-bromophenol-degrading, sulfate-reducing consortium. Starved, washed cultures were amended with 2-bromophenol plus sulfate, 2-bromophenol plus hydrogen, phenol plus sulfate, or phenol with no electron acceptor and were monitored for substrate use. In the presence of sulfate, 2-bromophenol and phenol were completely degraded. In the absence of sulfate, 2-bromophenol was dehalogenated and phenol accumulated. Direct terminal restriction fragment lengthpolymorphism fingerprinting of the 16S rRNA in the various subcultures indicated that phylotype 2BP-48 (a Desulfovibrio-like sequence) was responsible for the dehalogenation of 2-bromophenol. A stable coculture was established which contained predominantly 2BP-48 and a second Desulfovibrio-like bacterium (designated BP212 based on terminal restriction fragment lengthpolymorphism fingerprinting) that was capable of dehalogenating 2-bromophenol to phenol. Strain 2BP-48 in the coculture could couple reductive dehalogenation to growth with 2-bromophenol, 2,6-dibromophenol, or 2-iodophenol and lactate or formate as the electron donor. In addition to halophenols, strain 2BP-48 appears to use sulfate, sulfite, and thiosulfate as electron acceptors and is capable of simultaneous sulfidogenesis and reductive dehalogenation in the presence of sulfate.

The Malayan box turtle (Cuora amboinensis) (MBT) is a vulnerable and protected turtle species, but it is a lucrative item in the illegal wildlife trade because of its great appeal as an exotic food item and in traditional medicine. Although several polymerase chain reaction (PCR) assays to identify MBT by various routes have been documented, their applicability for forensic authentication remains inconclusive due to the long length of the amplicon targets, which are easily broken down by natural decomposition, environmental stresses or physiochemical treatments during food processing. To address this research gap, we developed, for the first time, a species-specific PCR-restriction fragment lengthpolymorphism (RFLP) assay with a very short target length (120 bp) to detect MBT in the food chain; this authentication ensured better security and reliability through molecular fingerprints. The PCR-amplified product was digested with Bfa1 endonuclease, and distinctive restriction fingerprints (72, 43 and 5 bp) for MBT were found upon separation in a microfluidic chip-based automated electrophoresis system, which enhances the resolution of short oligos. The chances of any false negative identifications were eliminated through the use of a universal endogenous control for eukaryotes, and the limit of detection was 0.0001 ng DNA or 0.01% of the meat under admixed states. Finally, the optimized PCR-RFLP assay was validated for the screening of raw and processed commercial meatballs, burgers and frankfurters, which are very popular in most countries. The optimized PCR-RFLP assay was further used to screen MBT materials in 153 traditional Chinese medicines of 17 different brands and 62 of them were found MBT positive; wherein the ingredients were not declared in product labels. Overall, the novel assay demonstrated sufficient merit for use in any forensic and/or archaeological authentication of MBT, even under a state of decomposition.

Full Text Available The Malayan box turtle (Cuora amboinensis (MBT is a vulnerable and protected turtle species, but it is a lucrative item in the illegal wildlife trade because of its great appeal as an exotic food item and in traditional medicine. Although several polymerase chain reaction (PCR assays to identify MBT by various routes have been documented, their applicability for forensic authentication remains inconclusive due to the long length of the amplicon targets, which are easily broken down by natural decomposition, environmental stresses or physiochemical treatments during food processing. To address this research gap, we developed, for the first time, a species-specific PCR-restriction fragment lengthpolymorphism (RFLP assay with a very short target length (120 bp to detect MBT in the food chain; this authentication ensured better security and reliability through molecular fingerprints. The PCR-amplified product was digested with Bfa1 endonuclease, and distinctive restriction fingerprints (72, 43 and 5 bp for MBT were found upon separation in a microfluidic chip-based automated electrophoresis system, which enhances the resolution of short oligos. The chances of any false negative identifications were eliminated through the use of a universal endogenous control for eukaryotes, and the limit of detection was 0.0001 ng DNA or 0.01% of the meat under admixed states. Finally, the optimized PCR-RFLP assay was validated for the screening of raw and processed commercial meatballs, burgers and frankfurters, which are very popular in most countries. The optimized PCR-RFLP assay was further used to screen MBT materials in 153 traditional Chinese medicines of 17 different brands and 62 of them were found MBT positive; wherein the ingredients were not declared in product labels. Overall, the novel assay demonstrated sufficient merit for use in any forensic and/or archaeological authentication of MBT, even under a state of decomposition.

Acenocoumarol is mainly catabolized by CYP2C9 isoform of cytochrome P450 (CYP) liver complex and exerts its anticoagulant effect through the inhibition of Vitamin K Epoxide Reductase (VKOR). The most important genetic polymorphisms which lead to an impaired enzymatic activity and therefore predispose to acenocoumarol sensitivity, are considered to be CYP2C9*2 (Arg144Cys), CYP2C9*3 (Ile359Leu) and VKORC1-1639G>A, respectively. In this study we compared the results of the PGXThrombo StripAssay kit (ViennaLab Diagnostics,Vienna, Austria) with direct DNA sequencing and in house Restriction Fragment LengthPolymorphisms (RFLP) for the detection of the aforementioned Single Nucleotide Polymorphisms (SNPs). The reverse hybridization StripAssay was found to be equally effective with RFLP and direct DNA sequencing for the detection of CYP2C9*2 and CYP2C9*3 polymorphisms, respectively. The comparison of the RFLP reference method with the reverse hybridization StripAssay for the detection of VKORC1-1639 G>A polymorphism showed that the reverse hybridization StripAsssay might misclassify some A/A homozygotes as heterozygotes. Optimization of the hybridization procedures may eliminate the extra low signal band observed in some samples at the reverse hybridization StripAssay and improve its diagnostic value.

Genetic variability of cultivated and wild barley, Hordeum vulgare ssp. vulgare and spontaneum, respectively, was assessed by RFLP analysis. The material consisted of 13 European varietes, single-plant offspring lines of eight land races from Ethiopia and Nepal, and five accessions of ssp....... spontaneum from Israel, Iran and Turkey. Seventeen out of twenty-one studied cDNA and gDNA probes distributed across all seven barley chromosomes revealed polymorphism when DNA was digested with one of four restriction enzymes. A tree based on genetic distances using frequencies of RFLP banding patterns...... an intermediate level. The proportion of gene diversity residing among,geographical groups (F-ST) varied from 0.19 to 0.94 (average 0.54) per RFLP pattern, indicating large diversification between geographical groups....

Full Text Available Bitter gourd plants showing symptoms of little leaf disease are prevalent in farmers’ fields in the Bangalore rural district, Karnataka state, India. Twenty leaf samples from different locations were collected to determine the etiology of the disease. Using PCR and phytoplasma 16S rRNA gene-specific universal primers, we observed positive amplification for the phytoplasma specific primers in five out of twenty samples. The amplified products were cloned, sequenced and nucleotide (NT sequence comparisons were made with the available phytoplasmas’ 16S rRNA gene NT sequences in the NCBI database. The 16S rRNA gene NT sequence of bitter gourd phytoplasma shared highest identity of 81.7-96.0% with ‘Candidatus Phytoplasma asteris’ (Ca. P. asteris 16Sr I group isolates from different parts of the world. This was supported by close clustering of phytoplasma of the current study with the Ca. P. asteris 16Sr I subgroup by phylogenetic analysis. The virtual restriction fragment lengthpolymorphism (RFLP pattern generated for the Phytoplasma from bitter gourd was in congruence with the in vitro RFLP pattern for the six enzymes. This was typical to Ca. P. asteris from the 16Sr I group. Further, virtual RFLP analysis with 11 more enzymes used for RFLP pattern prediction revealed differences only in the Mse I RFLP pattern, with a similarity coefficient of 0.91, which is less than the threshold similarity coefficient for a new subgroup. We propose that the phytoplasma detected in the present study that infects bitter gourd and causes littleleaf disease should be considered as a new subgroup of group 16Sr I (Ca. P. asteris. This is the first report of phytoplasma associated with littleleaf disease of bitter gourd from India.

Full Text Available Background: Campylobacter species are responsible for the majority of cases of food-borne gastroenteritis. The sources of the disease outbreaks are often contaminated water or milk, and consumption of undercooked poultry product is the main cause of sporadic campylobacteriosis cases. Objectives: The aims of this study were to determine the prevalence of Campylobacter gastroenteritis in children and to differentiate the interfering species using polymerase chain reaction (PCR and restriction fragment lengthpolymorphism (RFLP methods at the Bahonar hospital in Karaj, Iran. Patients and Methods: A total of 150 stool samples were collected from children under 10 years old during the summer of 2014. PCR was performed using genus- and species-specific primers and RFLP was done using AluI and TasI enzymes. Results: The results showed the amplification of 400 and 491 bp segments and Campylobacter contamination in 30 (20% samples; 5 out of 30 Campylobacter positive samples (16.66% were identified as C. jejuni, 20 (66.66% as C. coli, 3 (10% as C. jejuni and C. coli (mixed infection, and 2 (6.66% were identified as non-jejuni, non-coli Campylobacter using the PCR method. Following the evaluation of RFLP results, 7 positive samples (23.33% showed the electrophoretic pattern of C. jejuni, 21 (70% showed the electrophoretic pattern of C. coli, and 2 (6.6% showed both of the patterns and mixed contamination with jejuni and coli species. The results of digestion with TasI did not show any C. lari or C. upsaliensis patterns. Conclusions: The results of this study showed high percentage of Campylobacter contamination in the tested stool samples. The other surprising finding was the high rate of Campylobacter coli positive samples; the difference between the results of PCR using species-specific primers (hipo and asp and the RFLP method (electrophoretic patterns in some of the positive samples confirms the hypothesis of variations in nucleotide sequences of the

Full Text Available Abstract Background Linkage disequilibrium (LD mapping is commonly used to evaluate markers for genome-wide association studies. Most types of LD software focus strictly on LD analysis and visualization, but lack supporting services for genotyping. Results We developed a freeware called LD2SNPing, which provides a complete package of mining tools for genotyping and LD analysis environments. The software provides SNP ID- and gene-centric online retrievals for SNP information and tag SNP selection from dbSNP/NCBI and HapMap, respectively. Restriction fragment lengthpolymorphism (RFLP enzyme information for SNP genotype is available to all SNP IDs and tag SNPs. Single and multiple SNP inputs are possible in order to perform LD analysis by online retrieval from HapMap and NCBI. An LD statistics section provides D, D', r2, δQ, ρ, and the P values of the Hardy-Weinberg Equilibrium for each SNP marker, and Chi-square and likelihood-ratio tests for the pair-wise association of two SNPs in LD calculation. Finally, 2D and 3D plots, as well as plain-text output of the results, can be selected. Conclusion LD2SNPing thus provides a novel visualization environment for multiple SNP input, which facilitates SNP association studies. The software, user manual, and tutorial are freely available at http://bio.kuas.edu.tw/LD2NPing.

Twenty-nine lines with deletions in the Lr19 ('Indis') translocated chromosome segment were used to physically map Thinopyrum Restriction Fragment LengthPolymorphism (RFLP) loci as well as the Sr25 and Sdl loci. The relative distances between marker loci on the translocation were then calculated. The information was then used as an aid to characterize several recombined forms of the translocation. The data confirmed the reported homoeology between the Lr19 segment and chromosome arm 7DL of wheat. Also, it seems that the Lr19 translocation in 'Indis' is very similar to the Lr19 segment in the T4 source and that the former may not derive from Thinopyrum distichum. Near-isogenic lines of the recombined segments were derived and used to study their expression of leaf rust resistance. It became evident that only one potentially useful recombinant was obtained in an earlier attempt to induce allosyndetic pairing between the Lr19 translocation and 7DL of wheat. (author)

Integration of classical genetic markers, in particular mutants, onto the maize Restriction Fragment LengthPolymorphism (RFLP) map will provide the tools necessary to further our understanding of plant development and of complex traits. Initially integration was accomplished by visual alignment of common markers and sometimes involved the use of information from several different molecular maps to determine the relative placement of a single mutant. The maize core marker set was designed to provide a common set of markers which could be used for integration of map data. We have completed the mapping, of 56 mutants on chromosome one relative to the core marker set. Phenotypes included whole plant, seedling, and kernel effects and represented a variety of biological processes. Since these mutants were previously located to chromosome arm, mapping required the use of only seven markers per mutant to define the correct bin location. Two mistakes in marker order relative to the classical map were identified, as well as, six groups of mutants which require allelism testing. Placement of mutants and cDNAs into bins using, the core markers provides a necessary resource for identification of gene function in maize. (author)

Full Text Available Aim: An attempt has been made to study the toll-like receptors 4 (TLR4 gene polymorphism from cattle DNA to correlate with mastitis cows. Materials and Methods: In present investigation, two fragments of TLR4 gene named T4CRBR1 and T4CRBR2 of a 316 bp and 382 bp were amplified by polymerase chain reaction (PCR, respectively from Kankrej (22 and Triple cross (24 cattle. The genetic polymorphisms in the two populations were detected by a single-strand conformational polymorphism in the first locus and by digesting the fragments with restriction endonuclease Alu I in the second one. Results: Results showed that both alleles (A and B of two loci were found in all the two populations and the value of polymorphism information content indicated that these were highly polymorphic. Statistical results of χ2 test indicated that two polymorphism sites in the two populations fit with Hardy–Weinberg equilibrium (p˂0.05. Meanwhile, the effect of polymorphism of TLR4 gene on the somatic cell score (SCS indicated the cattle with allele a in T4CRBR1 showed lower SCS than that of allele B (p<0.05. Thus, the allele A might play an important role in mastitis resistance in cows. Conclusion: The relationship between the bovine mastitis trait and the polymorphism of TLR4 gene indicated that the bovine TLR4 gene may play an important role in mastitis resistance.

Full Text Available Research with aim to estimate genetic polymorphism at ACACA (Acetyl-coenzyme A carboxylase locus in Ettawa Crossbred goat wan its relationship with production traits was done at goat population in Batu, Lawang and Ampel Gading. 46 female goats were taken it’s blood sample to isolate the DNA and continue with PCR (Polymerase Chain Reaction and RFLP (Restricted Fragment LengthPolymorphism. PCR was used to amplify ACACA gene fragment in intron 3’ about 200 bp with primer F : 5’ – AGT GTA GAA GGG ACA GCC CAG C – 3’ and R : 5’ – GTG GAA TGA CAC ATG GAG AGG G – 3’; RFLP was used to test mutation of that fragment in particular place (point using restriction enzyme RSA1. Variables were alelles and genotypes composition in population, milk and fat content, and birth weight of kid. Result showed that (a genetic polymorphism at locus ACACA in three location was high that is 44,22 %, with allele frequency of G (p = 33 % and allele T (q = 67 %; (b no relationship between the high polymorphism with productive performance of goat in fat and protein content, and birth weight of kid. It was concluded that in goat population there was a high polymorphism at ACACA gene, and that polymorphism was not related to production.

In the genus Pinus the internal transcribed spacers (ITS1 and ITS2) and the 5.8s region of the nuclear ribosomal DNA are approximately 3000 bp in length. ITS1 is considerably longer than ITS2 and partial sequences of ITS1 indicate that this region is evolving rapidly and exhibits intraspecific variation. The ITS2 and 5.8s regions are relatively conserved. We surveyed restriction fragment length variability of PCR-amplified fragments (PCR-RFLP) of the ITS region in four populations (86 individuals) of Pinus rzedowskii, a pine endemic to western Michoacán, Mexico. Five of the restriction endonucleases assayed revealed variation, with a total of 13 variants, most of which were length mutations of 300-900 bp. A moderate degree of population differentiation was detected. The average diversity (Shannon's index) of ITS fragment size patterns was 1.19, with 34% of the variation due to differences among populations and 66% due to differences among individuals within populations. The same individuals were assayed for nine polymorphic isozymes, which gave diversity measures similar to those of each restriction endonuclease.

, were investigated by analysis of amplified fragment lengthpolymorphisms of the Bgl II and Mfe I restriction sites and by pulsed-field gel electrophoresis of a Bss HII digest of chromosomal DNA. Both methods allowed unambiguous differentiation of the analysed strains and showed similar discriminatory...... potential for the differentiation of M. hyosynoviae isolates. Concordant results obtained with the two whole-genome fingerprinting techniques evidence the considerable intraspecies genetic heterogeneity of M. hyosynoviae . Sixteen field strains of M. hyosynoviae and the type strain S16(T) were further...

Full Text Available The objective of this research was to identify growth hormone (GH genes genotype in selectedAceh cattle at Indrapuri’s Breeding and Forage Centre (IBFC of Aceh Cattle. Fourty one cattleconsisting of 21 male and 20 female cattle were used in this study. The genomic DNA was extractedfrom blood using Sambrook et al. (1989 methods. Polymerase Chain Reaction - Restriction FragmentLengthPolymorphism (PCR-RFLP and mehod of sequencing was used to detect MspI site on GH gene.Based on sequencing results, it can be concluded that all cattle were monomorphism. The frequency ofTT genotype were 1.00 and same as T allele frequency. The transition of C (cytosine into T (thymineon 1548 bp caused the lost of restriction site.

A polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP) assay developed for identification of five species of the Anopheles minimus Theobald group and a related mosquito species of the Myzomyia Series (Diptera: Culicidae) was applied to morphologically identified adult female specimens collected in Ratanakiri Province, north-eastern Cambodia. In addition to finding An. aconitus Dönitz, An. minimus species A and An. pampanai Büttiker & Beales, some specimens showed a new restriction banding pattern. Siblings of specimens that exhibited this new PCR-RFLP pattern were morphologically identified as An. culicifacies James sensu lato. Based on nucleotide sequences of the ribonuclear DNA internal transcribed spacer 2 region (ITS2) and the mitochondrial cytochrome oxidase I gene (COI), these specimens were recognized as An. culicifacies species B (sensu Green & Miles, 1980), the first confirmed record of the An. culicifacies complex from Cambodia. This study shows that the PCR-RFLP assay can detect species not included in the initial set-up and is capable of identifying at least seven species of the Myzomyia Series, allowing better definition of those malaria vector and non-vector anophelines in South-east Asia.

Temporal and spatial variation of communities of soil denitrifying bacteria at sites receiving mineral fertilizer (60 and 120 kg N ha-1 year-1) and cattle manure (75 and 150 kg N ha-1 year-1) were explored using terminal restriction fragment lengthpolymorphism (T-RFLP) analyses of PCR amplified...... nitrite reductase (nirK and nirS) gene fragments. The analyses were done three times during the year: in March, July and October. nirK gene fragments could be amplified in all three months, whereas nirS gene fragments could be amplified only in March. Analysis of similarities in T-RFLP patterns revealed...... a significant seasonal shift in the community structure of nirK-containing bacteria. Also, sites treated with mineral fertilizer or cattle manure showed different communities of nirK-containing denitrifying bacteria, since the T-RFLP patterns of soils treated with these fertilizers were significantly different...

We analyzed the relationship between Mink-S27 gene polymorphism and children with cardiac insufficiency. From April 2013 to April 2015, we enrolled 73 cases of children with cardiac insufficiency for this study, and all 73 were placed in the observation group. 76 normal cases were selected for the control group. Restriction fragment lengthpolymorphism (RFLP) was used to make polymorphism analysis of the Mink-S27. Our results showed no significant differences in Mink-S27 genotype and allele distribution in both observation and control groups (p>0.05). In lesion samples collected from children with cardiac insufficiency, we detected significant difference in AA, CC genotype frequency and allele frequency between the observation group and the control group (prelatively high. GNAS2 gene polymorphism was associated with the prevalence of cardiac insufficiency in children. And also the patients' condition was correlated to the frequency of different genotypes and alleles.

We developed a simple and reliable method to identify carnivore scats to species using PCR and RFLP of a portion of the mtDNA cytochrome b gene, which works for seven of the most common carnivores in western North America. We identified a short (196 bp) polymorphic region of cytochrome b which would

Full Text Available Polymerase chain reaction-amplified product lengthpolymorphism (PCR-APLP is one of the most convenient and reliable methods for single nucleotide polymorphism (SNP analysis. This method is based on PCR, but uses allele-specific primers containing SNP sites at the 3'-terminus of each primer. To use this method at least two allele-specific primers and one "counter-primer", which serves as a common forward or reverse primer of the allele-specific primers, are required. The allele-specific primers have SNP sites at the 3'-terminus, and another primer should have a few non-complementary flaps at the 5'-terminus to detect SNPs by determining the difference of amplicon length by PCR and subsequent electrophoresis. A major disadvantage of the addition of a non-complementary flap is the non-specific annealing of the primer with non-complementary flaps. However, a design principle for avoiding this undesired annealing has not been fully established, therefore, it is often difficult to design effective APLP primers. Here, we report allele-specific primers with an inosine chain at the 5'-terminus for PCR-APLP analysis. This unique design improves the competitiveness of allele-specific primers and the reliability of SNP analysis when using the PCR-APLP method.

One hundred and ninety five subjects of the Semai group of Orang Asli in peninsular Malaysia were examined for the distribution of Ag(c/g) and Ag(al/d) RFLPs of the apoB gene. Regions of apoB gene corresponding to nt 421 and 1981 representing these two Ags were amplified by polymerase chain reaction using primers of published sequences. Thr{sub 71} to Ile (Ag c/g) was detected as an ApaL I RFLP and Val{sub 591} to Ala (Ag al/d) by Alu I RFLP. DNA fragments were separated by 4% agarose gel electrophoresis and photographed over a UV transilluminator. The frequencies of Ag(d) (absence of ApaL I site) and Ag(d) (presence of Alu I site) were found to be 0.13 and 0.14, respectively, in the Orang Asli compared to frequencies of 0.30 and 0.45 in the Caucasian population. Distribution of the genotypes of these two polymorphisms was at Hardy-Weinberg equiilibrium.

Aflatoxins (AFs) represent the most important single mycotoxin-related food safety problem in developed and developing countries as they have adverse effects on human and animal health. They are produced mainly by Aspergillus flavus and A. parasiticus. Both species have different aflatoxinogenic profile. In order to distinguish between A. flavus and A. parasiticus, gene-specific primers were designed to target the intergenic spacer (IGS) for the AF biosynthesis genes, aflJ and aflR. Polymerase chain reaction (PCR) products were subjected to restriction endonuclease analysis using BglII to look for restriction fragment lengthpolymorphisms (RFLPs). Our result showed that both species displayed different PCR-based RFLP (PCR-RFLP) profile. PCR products from A. flavus cleaved into 3 fragments of 362, 210, and 102 bp. However, there is only one restriction site for this enzyme in the sequence of A. parasiticus that produced only 2 fragments of 363 and 311 bp. The method was successfully applied to contaminated grapes samples. This approach of differentiating these 2 species would be simpler, less costly, and quicker than conventional sequencing of PCR products and/or morphological identification. (author)

The tuberous roots of Pueraria candollei (White Kwao Khruea), Butea superba (Red Kwao Khruea) and Mucuna collettii (Black Kwao Khruea), which belong to the family Leguminosae, are used as rejuvenating herbs in traditional Thai medicine. Although all of these species have an indication for rejuvenation, each differs in its medicinal properties. Two varieties of P. candollei, var. mirifica and var. candollei, affect females, whereas B. superba and M. collettii exhibit effects on males. However, the identification of these roots according to the name "Kwao Khruea" is confusing due to the similarity in their features. Polymerase chain reaction restriction fragment lengthpolymorphism (PCR-RFLP) was utilised to identify plant origin. The partial matK gene was amplified and subjected to restriction enzyme digestion with DdeI and TaqI. The restriction fragments generated differed in number and size. To test the reliability of the method, an admixture of the different Kwao Khruea species containing equal amounts of DNA was tested. The results showed combined restriction patterns, and each species could be detected in the background of the others. The method was also used to authenticate eight different crude drugs sold as various types of Kwao Khruea in Thai markets. The results showed that the misidentification of commercial drugs remains a problem in crude drug markets. The PCR-RFLP analysis developed here provides a simple and accurate discrimination of these rejuvenating "Kwao Khruea" species.

Full Text Available The aim of the present study was to compare polymerase chain reaction (PCR-based methods - spoligotyping and mycobacterial interspersed repetitive units (MIRU typing - with the gold-standard IS6110 restriction fragment lengthpolymorphism (RFLP analysis in 101 isolates of Mycobacterium tuberculosis to determine the genetic diversity of M. tuberculosis clinical isolates from Delhi, North India. Spoligotyping resulted in 49 patterns (14 clusters; the largest cluster was composed of Spoligotype International Types (SITs26 [Central-Asian (CAS1-Delhi lineage], followed by SIT11 [East-African-Indian (EAI 3-Indian lineage]. A large number of isolates (75% belonged to genotypic lineages, such as CAS, EAI and Manu, with a high specificity for the Indian subcontinent, emphasising the complex diversity of the phylogenetically coherent M. tuberculosis in North India. MIRU typing, using 11 discriminatory loci, was able to distinguish between all but two strains based on individual patterns. IS6110-RFLP analysis (n = 80 strains resulted in 67 unique isolates and four clusters containing 13 strains. MIRUs discriminated all 13 strains, whereas spoligotyping discriminated 11 strains. Our results validate the use of PCR-based molecular typing of M. tuberculosis using repetitive elements in Indian isolates and demonstrate the usefulness of MIRUs for discriminating low-IS6110-copy isolates, which accounted for more than one-fifth of the strains in the present study.

Twelve isolates of Acanthamoeba spp. assigned to either A. castellanii or A. polyphaga, and type strains of A. culbertsoni, A. healyi, A. palestinensis, and A. astronyxis were examined by restriction fragment lengthpolymorphism (RFLP) of a conserved region of small subunit ribosomal RNA gene (ssu rDNA) amplified by polymerase chain reaction (PCR). The PCR products of the isolates measured approximately 910-930 bp, except for that of A. astronyxis which was extraordinarily long, approximately 1,170 bp. Average of estimated sequence divergence of the amplified DNA among the isolates assigned to A. castellaii was 9.8% whereas that among the isolates assigned to A. polyphaga 9.6%. The maximum intraspecific sequence divergence among the isolates assigned to A. castellanii was observed between the Chang and Ma strains (17.3%) while that among the isolates assigned to A. polyphaga was observed between KA/S3 and KA/S7 strains (16.1%). The both maximum sequence divergences were much greater than the minimum interspecific sequence divergence between A. castellanii and A. polyphaga (2.6%) which appeared between the Castellani (or CCAP 1501/2 g) and KA/S3 strains. The PCR-RFLP patterns of A. culbertsoni, A. healyi, A. palestinensis, and A. astronyxis were quite diverse from one another and from those of isolates assigned to either A. castellanii or A. polyphaga. It is suggested that taxonomic validity of the isolates assigned to either A. castellanii or A. polyphaga should be reevaluated.

Full Text Available Abstract Potato virus M (PVM, Carlavirus is considered to be one of the most common potato viruses distributed worldwide. Sequences of the coat protein (CP gene of several Canadian PVM isolates were determined. Phylogenetic analysis indicated that all known PVM isolates fell into two distinct groups and the isolates from Canada and the US clustered in the same group. The Canadian PVM isolates could be further divided into two sub-groups. Two molecular procedures, reverse transcription - polymerase chain reaction (RT-PCR and restriction fragment lengthpolymorphism (RFLP were developed in this study for the detection and identification of PVM in potato tubers. RT-PCR was highly specific and only amplified PVM RNA from potato samples. PVM RNAs were easily detected in composite samples of 400 to 800 potato leaves or 200 to 400 dormant tubers. Restriction analysis of PCR amplicons with MscI was a simple method for the confirmation of PCR tests. Thus, RT-PCR followed by RFLP analysis may be a useful approach for screening potato samples on a large scale for the presence of PVM.

Entamoeba histolytica and Entamoeba dispar are two morphologically indistinguishable human protozoan parasites that are genetically distinct species. The potential invasive pathogenic Entamoeba histolytica and non-invasive parasites Entamoeba dispar can be differentiated by molecular and other methods. We used Polymer Chain Reaction (PCR) to determine the ratio of two species in a population in central Iran.Human stool samples(n=12 148) were randomly collected in Tehran and Karaj and examined for E.histolytica/E.dispar cysts with direct and formalin-ether methods.Eighty -seven (0.7%) cases were positive, of which 49 (62.8%) isolates were successfully cultured in Robison's medium. A pair of oligonucleotide primers designed from sequence data for genomic DNA coding the 30-KD surface antigen of E.histolytica/E.dipar was used to amplify a 374 base-pair (bp) fragment. The restriction fragment lengthpolymorphism (RFLP) pattern obtained from a standard E.histolytica isolate had had two fragments (219 bp and 155bp), but the standard isolate of E. disparshowed three fragments (155, 152 and 67bp ). Differential diagonosis of 49 isolates of E. histolytica/ E. dispar from Tehran and Karaj using PCR-RFLP revealed that 46(93.9%) were E. were E. dispar while only 2(4.1%) were E. histolytica .One person (2%) had a mixed infection and showed both patterns The differential diagonosis of the potentially pathogenic parasite E.histolytica from the non-pathogenic E. dispar is of clinical and epidemiological importance. This study demonstrated E. dispar is much more prevalent than E.histolytica among the c yst passersin Tehran and Kraj in Central Iran. (author)

Full Text Available Background: Due to the predisposing conditions in patients with cystic fibrosis (CF caused by defective mucociliary clearance facilitating colonization and invasion with Candida species has dramatically increased. Traditional methods for identifying problems are imminent and time-consuming. Therefore, molecular techniques utilizing amplification of target DNA provide quick and precise methods for the diagnosis and identification of Candida species. Therefore, the aim of the current study was to identify the most medically common isolated Candida species from the air way of CF patients by PCR-RFLP and amplification of HWP1 gene. Materials and Methods: A total of 42 CF patients presenting symptoms who referred to pediatric respiratory diseases research center were screened for the presence of Candida spp. The isolates initially were phenotypically identified and confirmed by molecular approaches based on restriction fragment lengthpolymorphism ( PCR -RFLP for the discrimination of C. albicans of non-albicans and the amplification of HWP1 gene for the discrimination of C. albicans from C. dubliniensis and C. africana was conducted. Results: The results show that C. albicans was the most frequently isolated species (83.8% followed by non-albicans included C. parapsilosis (7.1%, C. glabrata (3.2%, and C. tropicalis (3.2%. The restriction patterns of each Candida species were perfectly specific. Since MspI could not discriminate between the three morphological related species, C. albicans, C. dubliniensis and C. africana, we used PCR amplification of HWP1 gene, which (7.1% species from C. albicans identified as C. dubliniensis, however C. africana strains were not found. Conclusion: The present study found that C. albicans as predominant species wereisolated from the CF patients. It could be concluded that molecular diagnostic methods are reliable and would be useful for the identification of medically important Candida species in clinical samples

Full Text Available A study was conducted to identify the polymorphism in the intron 3 of the Growth Hormone (GH gene and also to evaluate the association of the GH gene polymorphism with growth parameters and dressing percentage in the Sumba Ongole (SO cattle. A total of 267 individual DNA samples were used in the Polymerase Chain Reaction-Restriction Fragment LengthPolymorphism (PCR-RFLP analysis. The SO cattle growth parameters data (n=44 including birth weight (BW, weaning weight at 205 days of age (WW205, yearling weight at 365 days of age (YW365 and also dressing percentage (DP (n=122 were investigated in this study. There were three genotypes (AA, AB, and BB of the GH gene based on the PCR-RFLP analysis with allele frequency was 0.87 and 0.13 for A allele and B allele respectively. The highest genotype frequency in the SO cattle is AA (0.76 and the lowest is BB (0.02. The Heterozygosity Observed (Ho value in the SO cattle population is 0.23 and Polymorphism Information Content (PIC value is 0.20. Therefore, the genetic diversity in the SO cattle based on the GH gene polymorphism is quite low. There is no association (P>0.05 in BW, WW205, YW365, and DP with genotypes of the GH gene. As the result, the GH gene in this study cannot be used as a genetic marker in the SO cattle breeding program.

From the study of 52 families and 15 homozygous typing cells, 234 MHC complement haplotypes were characterized for features in the DNA of the complotype region: C2/Sst I (2.75, 2.70, 2.65, and 2.40 kb), BF/Taq I (6.6 and 4.5 kb), C4 5'/Bgl II (15 and 4.5 kb), C4 5'/Taq I (7.0, 6.4, 6.0 and 5.4 kb) and C4 3'/Xba I/BamH I (11 and 4 + 7 kb) restriction fragment lengthpolymorphisms (RFLP's), by the presence or absence of C4A, C4B, CYP21A and CYP21B genes and by duplications. Nineteen (of over 1000 theoretically possible) complotype-RFLP constellations (CRC's) were found. The 9 CRC's with two C4 and CYP21 genes were designated A through I. CRC's Bdup and Ddup were like B and D but had duplicated C4B-CYP21B genes. The remaining CRC's had deletions of C4 and/or CYP21 genes and were designated Bdel, Cdel and the like. Individual complement alleles and complotypes were nor randomly distributed among the CRC's. Some complotypes, such as SC01, SC02 and FIC30, were restricted to only 1 CRC; others, such as SC31, FC31, and SC30, were found in several CRC's. Some of the CRC's contained a single complotype, others contained several. Remarkably, there are about 30 CRC-specified complotypes with frequencies of .01 or higher and 14 of .02 or higher. A number of evolutionary origins of complement alleles and complotypes are suggested by the relationships among CRC's. Approximate normal frequencies of the undeleted CRC's were A = .27, B = .19, Bdup = .02, C = .17, D = .07, Ddup = .02, E = .06, F = .05, and G = .02. Thus, CRC's without deletions accounted for 88% of normal complotypes. Since the frequency of Bdel, with a deletion of C4A, was .12, 10 CRC's accounted for all observed normal caucasian MHC haplotypes.

Functional polymorphisms in genes encoding enzymes involved in folate metabolism might modulate head and neck carcinoma risk because folate participates in DNA methylation and synthesis. We therefore conducted a case-control study of 853 individuals (322 head and neck cancer cases and 531 non-cancer controls) to investigate associations among MTHFR C677T and MTHFR A1298C polymorphisms and head and neck squamous cell carcinoma risk. Interactions between these two polymorphisms and risk factors and clinical histopathological parameters were also evaluated. The polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP) technique was used to genotype the polymorphisms and Chi-square test and multiple logistic regression were used for statistical analyses. The variables age≥49 years, male gender, tobacco habits and alcohol consumption, MTHFR 1298 AC or CC genotypes, combined genotypes with two or more polymorphic alleles and 677T and 1298C polymorphic alleles were associated with increased risk for this disease (PA1298C polymorphism was more frequent in patients with oral cavity as primary site (PA1298C polymorphism has higher risk for this disease.

Full Text Available Abstract Background Improvement of efficiency and economic returns is an important goal in dairy farming, as in any agricultural enterprise. The primary goal of dairy industry has been to identify an efficient and economical way of increasing milk production and its constituents without increasing the size of the dairy herd. Selection of animals with desirable genotypes and mating them to produce the next generation has been the basis of livestock improvement and this would continue to remain the same in the coming years. The use of polymorphic genes as detectable molecular markers is a promising alternative to the current methods of trait selection once these genes are proven to be associated with traits of interest in animals. The point mutations in exon IV of bovine β-Lactoglobulin gene determine two allelic variants A and B. These variants were distinguished by Polymerase Chain Reaction and Restriction Fragment LengthPolymorphism (PCR-RFLP analysis in two indigenous Bos indicus breeds viz. Sahiwal and Tharparkar cattle. DNA samples (228 in Sahiwal and 86 in Tharparkar were analyzed for allelic variants of β-Lactoglobulin gene. Polymorphism was detected by digestion of PCR amplified products with Hae III enzyme, and separation on 12% non-denaturing gels and resolved by silver staining. Results The allele B of β-Lactoglobulin occurred at a higher frequency than the allele A in both Sahiwal and Tharparkar breeds. The genotypic frequencies of AA, AB, and BB in Sahiwal and Tharparkar breeds were 0.031, 0.276, 0.693 and 0.023, 0.733, 0.244 respectively. Frequencies of A and B alleles were 0.17 and 0.83, and 0.39 and 0.61 in Sahiwal and Tharparkar breeds respectively. The Chi-square test results (at one degree of freedom at one per cent level revealed that the Tharparkar population was not in Hardy-Weinberg equilibrium as there was a continuous migration of animals in the herd studied, where as, the results are not significant for the Sahiwal

Full Text Available Background. Recent data have suggested that polymorphisms in the length of the polyalanine tract (polyA of FOXE1 gene may act as a susceptibility factor for thyroid dysgenesis. The main purpose of this study was to investigate the influence of polyA of FOXE1 gene on the risk of thyroid dysgenesis. Method. A case-control study was conducted in a sample of 90 Brazilian patients with thyroid dysgenesis and 131 controls without family history of thyroid disease. Genomic DNA was isolated from peripheral blood samples and the genotype of each individual was determined by automated sequencing. Results. More than 90% of genotypes found in the group of patients with thyroid dysgenesis and in controls subjects were represented by sizes 14 and 16 polymorphisms in the following combinations: 14/14, 14/16, and 16/16. Genotypes 14/16 and 16/16 were more frequent in the control group, while genotype 14/14 was more frequent in the group of patients with thyroid dysgenesis. There was no difference between agenesis group and control group. Genotype 14/14 when compared to genotypes 14/16 and 16/16A showed an association with thyroid dysgenesis. Conclusion. PolyA of FOXE1 gene alters the risk of thyroid dysgenesis, which may explain in part the etiology of this disease.

The experiment was carried out at the Department of Horticulture, Ubon Ratchathani University, Ubon Ratchathani province, Northeast Thailand during June 2002 to May 2003 aims to identify DNA fingerprints of thirty papaya cultivars with the use of Amplified Fragment LengthPolymorphisms (AFLP) technique. Papaya cultivars were collected from six different research centers in Thailand. Papaya plants of each cultivar were grown under field conditions up to four months then leaf numbers 2 and 3 of each cultivar (counted from top) were chosen for DNA extraction and the samples were used for AFLP analysis. Out of 64 random primers being used, 55 pairs gave an increase in DNA bands but only 12 pairs of random primers were randomly chosen for the final analysis of the experiment. The results showed that AFLP markers gave Polymorphic Information Contents (PIC) of three ranges i.e., AFLP markers of 235 lied on a PIC range of 0.003-0.05, 47 for a PIC range of 0.15-0.20 and 12 for a PIC range of 0.35-0.40. The results on dendrogram cluster analysis revealed that the thirty papaya cultivars were classified into six groups i.e., (1) Kaeg Dum and Malador (2) Kaeg Nuan (3) Pakchong and Solo (4) Taiwan (5) Co Coa Hai Nan and (6) Sitong. Nevertheless, in spite of the six papaya groups all papaya cultivars were genetically related to each other where diversity among the cultivars was not significantly found.

Sex determination is important in archeology and anthropology for the study of past societies, cultures, and human activities. Sex determination is also one of the most important components of individual identification in criminal investigations. We developed a new method of sex determination by detecting a single-nucleotide polymorphism in the amelogenin gene using amplified product-lengthpolymorphisms in combination with sex-determining region Y analysis. We particularly focused on the most common types of postmortem DNA damage in ancient and forensic samples: fragmentation and nucleotide modification resulting from deamination. Amplicon size was designed to be less than 60 bp to make the method more useful for analyzing degraded DNA samples. All DNA samples collected from eight Japanese individuals (four male, four female) were evaluated correctly using our method. The detection limit for accurate sex determination was determined to be 20 pg of DNA. We compared our new method with commercial short tandem repeat analysis kits using DNA samples artificially fragmented by ultraviolet irradiation. Our novel method was the most robust for highly fragmented DNA samples. To deal with allelic dropout resulting from deamination, we adopted “bidirectional analysis,” which analyzed samples from both sense and antisense strands. This new method was applied to 14 Jomon individuals (3500-year-old bone samples) whose sex had been identified morphologically. We could correctly identify the sex of 11 out of 14 individuals. These results show that our method is reliable for the sex determination of highly degenerated samples. PMID:28052096

Full Text Available Sex determination is important in archeology and anthropology for the study of past societies, cultures, and human activities. Sex determination is also one of the most important components of individual identification in criminal investigations. We developed a new method of sex determination by detecting a single-nucleotide polymorphism in the amelogenin gene using amplified product-lengthpolymorphisms in combination with sex-determining region Y analysis. We particularly focused on the most common types of postmortem DNA damage in ancient and forensic samples: fragmentation and nucleotide modification resulting from deamination. Amplicon size was designed to be less than 60 bp to make the method more useful for analyzing degraded DNA samples. All DNA samples collected from eight Japanese individuals (four male, four female were evaluated correctly using our method. The detection limit for accurate sex determination was determined to be 20 pg of DNA. We compared our new method with commercial short tandem repeat analysis kits using DNA samples artificially fragmented by ultraviolet irradiation. Our novel method was the most robust for highly fragmented DNA samples. To deal with allelic dropout resulting from deamination, we adopted "bidirectional analysis," which analyzed samples from both sense and antisense strands. This new method was applied to 14 Jomon individuals (3500-year-old bone samples whose sex had been identified morphologically. We could correctly identify the sex of 11 out of 14 individuals. These results show that our method is reliable for the sex determination of highly degenerated samples.

An encapsulation/dehydration procedure was developed for Euglena gracilis Klebs as a 'model alga' to examine various cryoprotective regimes combined with controlled rate cooling to cryopreserve other Euglenoid taxa. Cryoprotective variables were optimised to enable reproducible growth following a combination of alginate encapsulation, sucrose osmotic dehydration, air desiccation, methanol treatment, cooling to -40 degrees C and plunging into liquid nitrogen (LN). Amplified Fragment LengthPolymorphism (AFLP) analysis was adapted to: (i) verify algal identity by discriminating between different Euglenoids and (ii) examine the genetic stability of algal cultures prior to various stages of cryoprotective treatments and following exposure to LN. AFLPs were highly reproducible (> 99%) as reliable diagnostic markers, where a single DNA fragment change accounted for -0.4% of the detectable variation in an AFLP pattern. AFLP changes were detected in cryoprotective treatments following LN exposure. Successive stages of the dehydration and desiccation treatments did not accumulate AFLP changes indicating these are random events.

health. The remaining 30 groups contained isolates from humans, chickens and associated food products, cattle, sheep, turkeys, ostriches and/or dogs. Strains assigned to serotypes 2, 6/7, 11 and 12 formed major clusters at the 77.6% S-level. Most other serotypes did not form homogeneous clusters......Aims: To use amplified fragment lengthpolymorphism (AFLP) analysis to evaluate the genetic relatedness among 254 Campylobacter jejuni reference and field strains of diverse origin representing all defined 'Penner' serotypes for this species. Methods and Results: Field strains (n = 207) from human...... diarrhoea and diverse animal and environmental sources were collected mainly through a National surveillance programme in Denmark and serotyped by use of the established 'Penner' scheme. Genetic relationships among these isolates, and the archetypal serotype reference strains, were assessed by numerical...

Full Text Available Aim. Autism is a subgroup of autism spectrum disorders, classified as a heterogeneous neurodevelopmental disorder and symptoms occur in the first three years of life. The etiology of autism is largely unknown, but it has been accepted that genetic and environmental factors may both be responsible for the disease. Recent studies have revealed that the genes involved in the folate/homocysteine pathway may be risk factors for autistic children. In particular, C677T polymorphism in the MTHFR gene as a possible risk factor for autism is still controversial. We aimed to investigate the possible effect of C677T polymorphism in a Turkish cohort. Methods. Autism patients were diagnosed by child psychiatrists according to DSM-IV and DSM-V criteria. A total of 98 children diagnosed as autistic and 70 age and sex-matched children who are nonautistic were tested for C677T polymorphism. This polymorphism was studied by using polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP methods. Results. MTHFR 677T-allele frequency was found to be higher in autistic children compared with nonautistic children (29% versus 24%, but it was not found statistically significant. Conclusions. We conclude that other MTHFR polymorphisms such as A1298C or other folate/homocysteine pathway genes may be studied to show their possible role in autism.

Androgens have an anti-proliferative effect on endometrial cells. Human androgen receptor (AR) gene contains two polymorphic short tandem repeats of GGC and CAG, and a single-nucleotide polymorphism on exon 1 that is recognized by the restriction enzyme, StuI. Prior studies have shown that the lengths of the CAG repeat are inversely and linearly related to AR activity and associated with endometrial cancer. However, little is known about the GGC repeat and the StuI polymorphism of the AR gene. Thus, we investigated whether these AR polymorphisms are risk factors for endometrial cancer. To test this hypothesis, the genetic distributions of these polymorphisms were investigated in blood samples from endometrial cancer patients and healthy controls. The allelic and genotyping profiles were analyzed by polymerase chain reaction (PCR), PCR-restriction fragment lengthpolymorphism (PCR-RFLP), and direct DNA sequencing, and analyzed statistically. The GGC repeat was significantly longer in endometrial cancer patients as compared to normal healthy controls. In general, an increased risk of endometrial cancer was found with increasing GGC repeat. The relative risk for the 17 GGC repeat was greater than 4, as compared to controls. However, the StuI polymorphism was not significantly different between patients and controls. The findings suggest that increased numbers of GGC repeat on the AR gene may be a risk factor for endometrial cancer

Bradykinin B2 receptor is involved in many processes, including the regulation of blood pressure and smooth muscle contraction, vasodilation, inflammation, edema, cell proliferation, pain. It is suggested that this receptor may be one of the factors that have cardioprotective and infarct-limiting effects. It is assumed that certain genetic variants in both coding and non-coding regions ofBDKRB2 gene may influence its expression. In the 3'-untranslated region of BDKRB2 exon 3 the minisatellite repeat B2-VNTR is located. B2-VNTR has previously been shown to affect the BDKRB2 mRNA stability. Therefore, it is important to perform the molecular genetic analysis of this minisatellite in patients with different forms of coronary heart disease in order to reveal possible associations between specific B2-VNTR alleles and certain clinical forms of coronary heart disease. In the present study, a comparative analysis of the allele and genotype frequencies of B2-VNTR was carried out in groups of healthy individuals and patients with two clinical forms of coronary heart disease (angina pectoris and myocardial infarction), ethnically Russian. The results of the B2-VNTR lengthpolymorphism analysis indicate that this tandem repeat may be attributed to a class of low polymorphic and non-hypervariable minisatellite. In all analyzed groups we revealed three B2-VNTR alleles, consisting of 43, 38 and 33 repeat units. Alleles of 43 and 33 repeats were major in all investigated groups. No statistically significant differences were found in the B2-VNTR allele and genotype frequencies between men and women in control group, and also between healthy men and men with angina pectoris and myocardial infarction. Thus, B2-VNTR lengthpolymorphism was not associated with these clinical forms of coronary heart disease in Russian men. However, we do not exclude the possibility of association between the B2-VNTR short alleles (38 and 33 repeats) and cardioprotective effects of bradykinin B2 receptor

Full Text Available Abstract Background The human multi-drug resistance gene (MDR1, which encodes the major trans-membrane transporter P-glycoprotein (P-gp, was found to be associated with susceptibility to cancer and response to chemotherapy. The C3435T Polymorphism of MDR1 gene was correlated with expression levels and functions of P-gp. Here, we studied the association between MDR1 C3435T polymorphism and susceptibility to Hodgkin lymphoma (HL and patient's response to ABVD chemotherapy regimen. Methods a total of 130 paraffin embedded tissue samples collected from HL patients were analyzed to identify the C3435T polymorphism. As a control group, 120 healthy subjects were enrolled in the study. The C3435T Polymorphism was genotyped by polymerase chain reaction and restriction fragment lengthpolymorphism (PCR-RFLP method. Data analysis was carried out using the statistical package SPSS version 17 to compute all descriptive statistics. Chi-square and Fisher exact tests were used to evaluate the genotype distribution and allele frequencies of the studied polymorphism. Results these studies revealed that the frequency of T allele was significantly higher in HL patients compared to the controls (P 0.05. Conclusions these results suggest that MDR1 C3435T polymorphism might play a role in HL occurrence; however this polymorphism is not correlated with the clinical response to ABVD.

Analysis of three genetic polymorphisms in Malaysian essential hypertensive and type 2 diabetic subjects. ... Genotyping of all the three polymorphisms was performed by PCR-RFLP method with the respective primers and restriction enzymes. The genotypic and allelic frequencies of the respective polymorphisms of the ...

Inflammatory bowel diseases (IBD) are disorders originated from immune disturbances. The AIM OF THE STUDY was to evaluate the association between the -2518 A/G MCP-1 polymorphism and the risk of IBD development. Genotypes were determined by polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP) method. Study group consisted of 197 subjects with IBD (120 with ulcerative colitis and 77 with Crohn's disease) as well as 210 healthy controls. The presence of the -2518 G/G MCP-1 genotype in the investigated groups seems to be connected with higher risk of inflammatory bowel disease as well as Crohn's disease only (OR 2.26; 95% CI 1.44-3.54 and OR 2.08; 95% CI 1.21-3.46, respectively). Our data showed that the -2518 A/G MCP-1 polymorphism might be associated with the IBD occurrence and might be used as predictive factor of these diseases in a Polish population.

Full Text Available A collection of 356 isolates of Aspergillus spp. collected during 2006 and 2007 from grapevines in northern Italy were identified through Internal Transcribed Spacer based Restriction Fragment LengthPolymorphism (ITS-RFLP and tested for ochratoxin A (OTA production. Restriction endonuclease digestion of the ITS products using the endonucleases HhaI, HinfI and RsaI, distinguished five different RFLPs. From each pattern, three samples were sequenced and the nucleotide sequences showed different species corresponding to Aspergillus niger, A. carbonarius, A. tubingensis, A. japonicus and A. aculeatus. By comparing the sequences of the ITS regions, also the uniseriate species A. japonicus and A. aculeatus could be differentiated by HinfI digestion of the ITS products. Among the aspergilli, A. niger was the major species associated with grapes during 2006 (57.4%, while A. carbonarius was the major species during 2007 (46.6%. All the strains of Aspergillus were tested for their ability to produce OTA on Yeast extract sucrose medium (YES, as it was tested as an optimal substrate for the evaluation of OTA production by black aspergilli. Out of 356 isolates, 63 (17.7% isolates produced OTA ranging from 0.05 to 3.0 µg mL−1. Most of the ochratoxigenic isolates were A. carbonarius (46 in both years, but also some strains of A. tubingensis (11 and A. japonicus (6 produced lower amounts of OTA.

To analyze quantitative trait loci (QTLs) affecting flooding tolerance and other physiological and morphological traits in Echinochloa crus-galli, a restriction fragment lengthpolymorphism (RFLP) map was constructed using 55 plants of the F(2) population ( E. crus-galli var. praticola x E. crus-galli var. formosensis). One hundred forty-one loci formed 41 linkage groups. The total map size was 1,468 cM and the average size of linkage groups was 35.8 cM. The average distance between markers was 14.7 cM and the range was 0-37.2 cM. Early comparisons to the genetic maps of other taxa suggest appreciable synteny with buffelgrass ( Pennisetum spp.) and sorghum ( Sorghum spp.). One hundred ninty-one F(2) plants were used to analyze QTLs of flooding tolerance, plant morphology, heading date, number of leaves, and plant height. For flooding tolerance, two QTLs were detected and one was mapped on linkage group 24. Other traits, including plant morphology, heading date, number of leaves, and plant height were highly correlated. Three genomic regions accounted for most of the mapped QTLs, each explaining 2-4 of the significant marker-trait associations. The high observed correlation between the traits appears to result from QTLs with a large contribution to the phenotypic variance at the same or nearby locations.

Inherited thrombophilic gene polymorphisms have been linked to the pathogenesis of venous thromboembolism (VTE). As there are very limited data of these polymorphisms in the Iranian population, we aimed to investigate the correlation between them and VTE in central Iran. Seventy-two unrelated VTE patients and 306 healthy control individuals were recruited for the study. Genotyping from venous blood with EDTA for the factor V Leiden (FVL), prothrombin (FII) G20210A, methylenetetrahydrofolate reductase (MTHFR) C677T and PLA2 polymorphism of platelet glycoprotein IIb/IIIa were undertaken by PCR-restriction fragment lengthpolymorphism (PCR-RFLP). A total of 57 investigated polymorphisms with a mean of 0.79 per individual and 151 with a mean of 0.49 were found in patients and controls, respectively (Pgenetic risk factor (P=0.007) and more recurrent events occurred in such patients. Patients with PLA2 polymorphism had more recurrent events than the other patients (P=0.02). Patients with more than one genetic risk factor and recurrent events were younger. The prevalence of these polymorphisms is different from some previously published data in other populations, but is consistent with some others. Higher prevalence of PLA2 polymorphism of GPIIa/IIIb in VTE patients is indicative of the impact of this polymorphism in the pathogenesis of VTE in this population. Because of the impact of coinheritance on the recurrence and the age of occurrence, such patients may need to be managed differently.

Full Text Available In order to assess the appropriateness of the use of internal transcribed spacer (ITS sequences for the study of population genetics of marine cyanobacteria, we amplified and cloned the 16S rRNA gene plus the 16S-23S ITS regions of six strains of Prochlorococcus and Synechococcus. We analyzed them by denaturing gradient gel electrophoresis (DGGE and terminal restriction fragment lengthpolymorphisms (T-RFLP. When using the standard application of these techniques, we obtained more than one band or terminal restriction fragment (T-RF per strain or cloned sequence. Reports in literature have suggested that these anomalies can result from the formation of secondary structures. Secondary structures of the ITS sequences of Prochlorococcus and Synechococcus strains were computationally modelled at the different temperatures that were used during the polymerase chain reaction (PCR. Modelling results predicted the existence of hairpin loops that would still be present at the extensión temperature; it is likely that these loops produced incomplete and single stranded PCR products. We modified the standard T-RFLP procedure by adding the labelled ITS primer in the last two cycles of the PCR reaction; this resulted, in most cases, in only one T-RF per ribotype. Application of this technique to a natural picoplankton community in marine waters off northern Chile, showed that it was possible to identify the presence, and determine the relative abundance, of several phylogenetic lineages within the genera Prochlorococcus and Synechococcus inhabiting the euphotic zone. Phylogenetic analysis of ITS sequences obtained by cloning and sequencing DNA from the same sample confirmed the presence of the different genotypes. With the proposed modification, T-RFLP profiles should therefore be suitable for studying the diversity of natural populations of cyanobacteria, and should become an important tool to study the factors influencing the genetic structure and

Full Text Available Background: Due to the lack of a suitable and economic test for the analysis of the polymorphism at codon 167, we developed a new PCR-RFLP technique, based on a modified forward primer (UT-HC167 MF-primer, to identify simultaneously the SNPs at codons 167 and 200 of isotype 1 β-tubu­lin gene of Haemonchus contortus.Methods: There already are several safe and easy methods for identification of point mutations at codons 198 and 200. Due to the lack of a reliable and easy method for the detection of the single nucleo­tide polymorphism (SNP at codon 167, we developed an innovative PCR-RFLP technique based on a modified forward primer (UT-HC167 MF-primer, in which the nucleotide T at the posi­tion 443 was substituted through a nucleotide A creating a restriction site for restriction endonuc­lease SnaB I in the nucleotide sequences including codon 167. A total of 138 adult male H. contortus were collected from three different geo-climatic areas of Iran. The isolated genomic DNA of each single worm was amplified by PCR using primers flanking codon 167. The PCR product (527 bp was then amplified by semi-nested PCR using the UT-HC167 MF-primer and the reverse primer achiev­ing a PCR product of 451 bp in length. This PCR product was subsequently digested with the restriction endonucleases SnaB I and TaaI for analysis of the mutations at codons 167 and 200, respec­tively.Results: All worms had two alleles encoding for phenylalanine (BZss homozygote for both codons.Conclusion: Using the UT-HC167 MF-primer and a suitable reverse primer designed upstream from codon 200, it is possible to amplify a PCR product which can be used for analysis of the SNPs at all three mentioned codons using RFLP.

Full Text Available Introduction & Objective: Periodontitis is a multifactorial disease in which host immune system and genetic factors have an important role in its pathogenesis. Genetic polymorphisms in cytokines and their receptors have been proposed as potential markers for periodontal diseases. The aim of the present study was to evaluate whether IL-4R gene polymorphism is associated with chronic periodontitis (CP or not? Materials & Methods: In this cross sectional study ninety non smoker patients (61 women and 29 men with chronic periodontitis were selected according to established criteria. They were categorized into three groups according to their clinical attachment level (CAL. Mutation at position 375(alanine/glutamine, 411(leucine/serine, 478(serine/proline, 406 (arginine/ cysteine in the IL-4R gene was detected by a polymerase chain reaction-restriction fragment lengthpolymorphisms (PCR-RFLP method.Results: The distribution of mutations for IL-4 polymorphism at amino acids 375 (P=0.41, 411(P=0.22, 478(P=0.17, 406(P=0.77 were not significantly different among mild, moderate and sever chronic periodontitis patients. Conclusion: This study suggests that there is no correlation between IL-4R polymorphism of chronic periodontitis.(Sci J Hamadan Univ Med Sci 2011;18(3:63-69

Aphids are among pests of economic importance throughout the world. Together with transmitting plant viruses, aphids are capable of inflicting severe crop production losses. They also excrete honeydew that favours the growth of sooty mold which reduces the quality of vegetables and fruits and hence their market values. Rapid and accurate identification of aphids to the species level is a critical component in effective pest management and plant quarantine systems. Even though morphological taxonomy has made a tremendous impact on species-level identifications, polymorphism, morphological plasticity and immature stages are among the many challenges to accurate identification. In addition, their small size, presence of cryptic species and damaged specimens dictate the need for a strategy that will ensure timely and accurate identification. In this study, polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP)-based on mitochondrial cytochrome c oxidase subunit I gene and DNA barcoding were applied to identify different aphid species collected from different agro-ecological zones of Kenya. Three restriction enzymes RsaI, AluI and Hinf1 produced patterns that allowed unambiguous identification of the species except Aphis craccivora and Aphis fabae. Analyses of the barcode region indicated intraspecific and interspecific sequence divergences of 0.08 and 6.63%, respectively. DNA barcoding identified all species, including the morphologically indistinguishable A. craccivora and A. fabae and separated two subspecies of A. fabae. Based on these results, both PCR-RFLPs and DNA barcoding could provide quick and accurate tools for identification of aphid species within Aphididae subsequently aiding in effective pest management programmes and enhance plant quarantine systems.

This case-control study was planned to investigate the possible role of methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C polymorphisms as a risk factor for the development of acute lymphoblastic leukemia (ALL) in a cohort of Egyptian children. Typing of MTHFR C677T and A1298C polymorphisms was done using restriction fragment lengthpolymorphism (RFLP) for 100 children with ALL and 100 age- and sex-matched healthy controls. No significant differences were found between patients with ALL and controls for the frequency of MTHFR C677T and A1298C alleles, genotypes, combined genotypes or haplotypes. The C677T and A1298C genotype frequency was different from that in Korean and Chinese populations (p 0.5). Our findings suggest that MTHFR C677T and A1298C polymorphisms are unlikely to affect the development of childhood ALL in an Egyptian population from Delta.

Several reports on science education suggest that students at all levels learn better if they are immersed in a project that is long term, yielding results that require analysis and interpretation. I describe a 12-wk laboratory project suitable for upper-level undergraduates and first-year graduate students, in which the students molecularly locate and map a gene from Drosophila melanogaster called dusky and one of dusky's mutant alleles. The mapping strategy uses restriction fragment lengthpolymorphism analysis; hence, students perform most of the basic techniques of molecular biology (DNA isolation, restriction enzyme digestion and mapping, plasmid vector subcloning, agarose and polyacrylamide gel electrophoresis, DNA labeling, and Southern hybridization) toward the single goal of characterizing dusky and the mutant allele dusky(73). Students work as individuals, pairs, or in groups of up to four students. Some exercises require multitasking and collaboration between groups. Finally, results from everyone in the class are required for the final analysis. Results of pre- and postquizzes and surveys indicate that student knowledge of appropriate topics and skills increased significantly, students felt more confident in the laboratory, and students found the laboratory project interesting and challenging. Former students report that the lab was useful in their careers.

Full Text Available Loss of heterozygosity (LOH is a common event in malignant cells. In this work we introduce a new approach to identify patients with loss of heterozygosity in the HLA region either at first diagnosis or after HLA mismatched allogeneic HSCT. Diagnosis of LOH requires a high purity of recipient target cells. FACS is time consuming and also frequently prevented by rather nonspecific or unknown immune phenotype. The approach for recipient cell enrichment is based on HLA targeted complement-dependent cytotoxicity (CDC. Relative fluorescent quantification (RFQ analysis of HLA intron lengthpolymorphisms then allows analysis of HLA heterozygosity. The approach is exemplified in recent clinical cases illustrating the detection of an acquired allele loss. As illustrated in one case with DPB1, distinct HLA loci in donor and patient were sufficient for both proof of donor cell removal and evaluation of allele loss in the patient's leukemic cells. Results were confirmed using HLA-B RFQ analysis and leukemia-associated aberrant immunophenotype (LAIP based cell sort. Both results confirmed suspected loss of HLA heterozygosity. Our approach complements or substitutes for FACS-based cell enrichment; hence it may be further developed as novel routine diagnostic tool. This allows rapid recipient cell purification and testing for loss of HLA heterozygosity before and after allogeneic HSCT in easily accessible peripheral blood samples.

The soft rot bacteria Erwinia carotovora and Erwinia chrysanthemi are important pathogens of potato and other crops. However, the taxonomy of these pathogens, particularly at subspecies level, is unclear. An investigation using amplified fragment lengthpolymorphism (AFLP) fingerprinting was undertaken to determine the taxonomic relationships within this group based on their genetic relatedness. Following cluster analysis on the similarity matrices derived from the AFLP gels, four clusters (clusters 1 to 4) resulted. Cluster 1 contained Erwinia carotovora subsp. carotovora (subclusters 1a and 1b) and Erwinia carotovora subsp. odorifera (subcluster 1c) strains, while cluster 2 contained Erwinia carotovora subsp. atroseptica (subcluster 2a) and Erwinia carotovora subsp. betavasculorum (subcluster 2b) strains. Clusters 3 and 4 contained Erwinia carotovora subsp. wasabiae and E. chrysanthemi strains, respectively. While E. carotovora subsp. carotovora and E. chrysanthemi showed a high level of molecular diversity (23 to 38% mean similarity), E. carotovora subsp. odorifera, E. carotovora subsp. betavasculorum, E. carotovora subsp. atroseptica, and E. carotovora subsp. wasabiae showed considerably less (56 to 76% mean similarity), which may reflect their limited geographical distributions and/or host ranges. The species- and subspecies-specific banding profiles generated from the AFLPs allowed rapid identification of unknown isolates and the potential for future development of diagnostics. AFLP fingerprinting was also found to be more differentiating than other techniques for typing the soft rot erwinias and was applicable to all strain types, including different serogroups.

Full Text Available The freshwater snails Biomphalaria straminea, B. intermedia, B. kuhniana and B. peregrina, are morphologically similar; based on this similarity the first three species were therefore grouped in the complex B. straminea. The morphological identification of these species is based on characters such as vaginal wrinkling, relation between prepuce: penial sheath:deferens vas and number of muscle layers in the penis wall. In this study the polymerase chain reaction restriction fragment lengthpolymorphism technique was used for molecular identification of these molluscs. This technique is based on the amplification of the internal transcribed spacer regions ITS1 e ITS2 of the ribosomal RNA gene and subsequent digestion of these fragments by restriction enzymes. Six enzymes were tested: Dde I, Mnl I, Hae III, Rsa I, Hpa II e Alu I. The restriction patterns obtained with DdeI presented the best profile for separation of the four species of Biomphalaria. The profiles obtained with all the enzymes were used to estimate the genetic distances among the species through analysis of common banding patterns.

Several reports on science education suggest that students at all levels learn better if they are immersed in a project that is long term, yielding results that require analysis and interpretation. I describe a 12-wk laboratory project suitable for upper-level undergraduates and first-year graduate students, in which the students molecularly locate and map a gene from Drosophila melanogaster called dusky and one of dusky's mutant alleles. The mapping strategy uses restriction fragment lengthpolymorphism analysis; hence, students perform most of the basic techniques of molecular biology (DNA isolation, restriction enzyme digestion and mapping, plasmid vector subcloning, agarose and polyacrylamide gel electrophoresis, DNA labeling, and Southern hybridization) toward the single goal of characterizing dusky and the mutant allele dusky73. Students work as individuals, pairs, or in groups of up to four students. Some exercises require multitasking and collaboration between groups. Finally, results from everyone in the class are required for the final analysis. Results of pre- and postquizzes and surveys indicate that student knowledge of appropriate topics and skills increased significantly, students felt more confident in the laboratory, and students found the laboratory project interesting and challenging. Former students report that the lab was useful in their careers. PMID:21364104

5,10-Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in folate metabolism and plays a major role in DNA methylation. There are two popular MTHFR polymorphisms known as C677T and A1298C which are found to be involved in folate metabolism and lowering the enzyme activity, thus may be linked with cancer development. This study aims to look at the association of these polymorphisms in gallbladder cancer. Thirty patients each with gallbladder cancer, cholelithiasis, and normal gallbladder were genotyped for the above-given polymorphisms by PCR-restriction fragment lengthpolymorphism (RFLP) method. C677T MTHFR polymorphism was not associated (χ(2) = 2.44, p = 0.85) with an increased likelihood of having gallbladder cancer. A1298C was significantly associated (χ(2) = 28.87, p A1298C was significantly correlated with grade (r = 0.337, p A1298C polymorphism may be associated with risk of developing gallbladder cancer, and there is no association between C677T polymorphism and gallbladder cancer.

Full Text Available Introduction: Migraine is a complex, recurrent headache disorder that is one of the most common complaints in neurology practice. The role of various genes in its pathogenesis is being studied. We did this study to see whether an association exists between ACE gene I/D polymorphism and migraine in our region. Materials and Methods: The study included 100 patients diagnosed with migraine and 121 healthy controls. The study subject were age and gender matched. The analysis was based on Polymerase Chain Reaction (PCR and included following steps: DNA extraction from blood, PCR and Restriction Fragment LengthPolymorphism (RFLP. Results: Out of 100 cases, 69 were females and 31 were males. Fifty-seven were having migraine without aura and 43 had migraine with aura. 45 of the cases had II polymorphism, 40 had ID polymorphism and 15 had DD polymorphism in ACE gene. Conclusion: We were not able to find a statistically significant association between ACE gene I/D polymorphism with migraine. The reason for difference in results between our study and other studies could be because of different ethnicity in study populations. So a continuous research is needed in this regard in order to find the genes and different polymorphism that increase the susceptibility of Kashmiri population to migraine.

Folate metabolism dysfunctions can result in DNA hypomethylation and abnormal chromosome segregation. Two common polymorphisms of the methylenetetrahydrofolate reductase (MTHFR) encoding gene (C677T and A1298C) reduce MTHFR activity, but when associated with aneuploidy, the results are conflicting. Turner Syndrome (TS) is an interesting model for investigating the association between MTHFR gene polymorphisms and nondisjunction because of the high frequency of chromosomal mosaicism in this syndrome. To investigate the association of MTHFR gene C677T and A1298C polymorphisms in TS patients and their mothers and to correlate these polymorphisms with maternal risk of TS offspring. MTHFR C677T and A1298C polymorphisms were genotyped in 33 TS patients, their mothers and 15 healthy females with their mothers as controls using polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP) and sequencing technique. Genotype and allele frequencies of both C677T and A1298C were not significantly different between TS cases and controls. There were no significant differences in C677T genotype distribution between the TS mothers and controls (p=1). The MTHFR 1298AA and 1298AC genotypes were significantly increased in TS mothers Vs. control mothers (p=0.002). The C allele frequency of the A1298C polymorphism was significantly different between the TS mothers and controls (p=0.02). The association of A1298C gene polymorphism in TS patients was found to increase with increasing age of both mothers (p=0.026) and fathers (p=0.044) of TS cases. Our findings suggest a strong association between maternal MTHFR A1298C and risk of TS in Egypt.

Adenosine deaminase-1 (ADA1) regulates the concentration of adenosine as the main modulator of oocyte maturation. There is compelling evidence for the association of ADA1 gene polymorphisms with many diseases but the importance of ADA1 polymorphisms in polycystic ovary syndrome (PCOS) has not been studied before. This study investigates serum total ADA activity (tADA), ADA1 and ADA2 isoenzyme activities, and genotype and allele frequencies of G22A and A4223C polymorphisms in healthy and PCOS women. In this case-control study 200 PCOS patients and 200 healthy women were enrolled. Genomic DNA was extracted from whole blood and the PCR-RFLP technique was used to determine the G22A and A4223C variants. The genotype frequencies were calculated and the association between polymorphic genotypes and enzyme activities were determined. tADA activity was significantly lower in the PCOS group compared with the control group (27.76±6.0 vs. 39.63±7.48, respectively). PCOS patients also showed reduced activity of ADA1 and ADA2. PCOS was not associated with G22A polymorphism whereas AA, AC, and CC genotypes of A4223C polymorphism were found distributed differently between the control and the PCOS women where the C allele showed a strong protective role for PCOS (odds ratio=1.876, p=0.033). The present study for the first time showed that lower ADA activity may be involved in pathogenesis of PCOS by maintaining a higher concentration of adenosine affecting follicular growth. As a novel finding, we also showed great differences in genotype distribution and allele frequencies of A4223C polymorphism between groups indicating a protective role for C allele against PCOS. AbbreviationsADA: adenosine deaminase PCOS: polycystic ovary syndrome PCR-RFLP: polymerase chain reaction-restriction fragment lengthpolymorphism tADA: total adenosine deaminase.

Full Text Available Abstract Background Previous studies suggested that genetic polymorphisms in the epidermal growth factor receptor (EGFR gene had been implicated in the susceptibility to some tumors and inflammatory diseases. EGFR has been recently implicated in vascular pathophysiological processes associated with excessive remodeling and atherosclerosis. Acute coronary syndrome (ACS is a clinical manifestation of preceding atherosclerosis. Our purpose was to investigate the association of the EGFR polymorphism with the risk of ACS. In this context, we analyzed the HER-1 R497K and EGFR intron 1 (CAn repeat polymorphisms in 191 patients with ACS and 210 age- and sex-matched controls in a Chinese population, using a polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP strategy and direct sequencing. Results There were significant differences in the genotype and allele distribution of R497K polymorphism of the EGFR gene between cases and controls. The Lys allele had a significantly increased risk of ACS compared with the Arg allele (adjusted OR = 1.49, 95% CI: 1.12–1.98, adjusted P = 0.006. However, no significant relationship between the number of (CAn repeats of EGFR intron 1 (both alleles P = 0.911. Considering these two polymorphisms together, there was no statistically significant difference between the two groups. Conclusion R497K polymorphism of the EGFR gene is significantly associated with the risk of ACS. Our data suggests that R497K polymorphism may be used as a genetic susceptibility marker of the ACS.

A novel and brief method of differentiating among horse (Equus caballus) and donkey (Equus asinus) and their hybrids (mule, E. asinus x E. caballus and hinny, E. caballus x E. asinus) with combined analysis of nuclear and mitochondrial gene polymorphism (CANMGP) was reported in the present report. A nuclear gene, protamine P1 gene of donkey was sequenced and compared with the known horse sequence from GenBank while a published equid mitochondrial gene, cytochrome b gene of donkey was compared with that of horse. In each of the two genes, a fixed nucleotide substitution within an exon that could be recognized by Dpn II restriction enzyme was found between the two species. Two pairs of primers were designed for amplifying the fragments within the two genes containing the informative nucleotide positions in 65 horses and 41 donkeys and 38 hybrids and conditions of polymerase chain reaction and restriction fragment lengthpolymorphism (PCR-RFLP) analysis were optimized. Horse, donkey and mule and hinny had their own specific cleavage patterns after the PCR-RFLP analysis was performed, which made it very easy to identify them from each other. As multiplex PCR can be conducted with the two pairs of primers and only one restriction enzyme is involved in PCR-RFLP analysis, the method described in the present study is a convenient way to identify horse and donkey and their hybrids. The idea involved in the method of CANMGP can be also used to differentiate other animal species or breeds and their hybrids.

Glutathione S-transferase (GST) is a xenobiotic metabolising enzyme (XME), which may modify susceptibility in certain ethnic groups, showing ethnic dependent polymorphism. The aim of this study was to determine GSTM1, GSTM3 and GSTT1 gene polymorphisms in a Malaysian population in Kuala Lumpur. Blood or buccal swab samples were collected from 137 Form II students from three schools in Wilayah Persekutuan Kuala Lumpur. Genotyping was done by polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP). Glutathione-S-transferase GSTM3 gene frequencies were 89% for AA, 10% for AB and 1% for BB. The gene frequencies for deleted GSTM1 and GSTT1 were 66% and 18% respectively. This study suggested that the Malay population is at risk for environmental diseases and provides the basis for gene-environment association studies to be carried out.

Glanzmann's thrombasthenia is a bleeding disorder caused by qualitative and/or quantitative defects of platelet membrane glycoprotein (GP) Ii b /III a complex. The disease is inherited in an autosomal recessive manner. The use of cDNA probes to study restriction fragment lengthpolymorphisms (RFLPs) in GPIII a gene is reported. A Taq I polymorphism was identified and this RFLP consisted of variant bands of 6.5 kb/4.0 and 2.5 kb with a frequency of 0.46/0.54 in Chinese population. The Taq I polymorphism was further localized by polymerase chain reaction (PCR) method to exon VIII of the GPIII a gene. In two Glanzmann's thrombasthenia families, the Taq I RFLP studied with both Southern blotting and PCR methods identified defective GPIII a gene inherited by patients, and determined the genotype of asymptomatic subjects. Analysis of this Taq I polymorphism by PCR method should be potentially useful in future for the carrier detection and prenatal diagnosis in Glanzmann's thrombasthenia families

Full Text Available Abstract Background Animal mycobacterioses are regarded as a potential zoonotic risk and cause economical losses world wide. M. avium subsp. hominissuis is a slow-growing subspecies found in mycobacterial infected humans and pigs and therefore rapid and discriminatory typing methods are needed for epidemiological studies. The genetic similarity of M. avium subsp. hominissuis from human and porcine origins using two different typing methods have not been studied earlier. The objective of this study was to compare the IS1245 RFLP pattern and MIRU-VNTR typing to study the genetic relatedness of M. avium strains isolated from slaughter pigs and humans in Finland with regard to public health aspects. Methods A novel PCR-based genotyping method, variable number tandem repeat (VNTR typing of eight mycobacterial interspersed repetitive units (MIRUs, was evaluated for its ability to characterize Finnish Mycobacterium avium subsp. hominissuis strains isolated from pigs (n = 16 and humans (n = 13 and the results were compared with those obtained by the conventional IS1245 RFLP method. Results The MIRU-VNTR results showed a discriminatory index (DI of 0,92 and the IS1245 RFLP resulted in DI 0,98. The combined DI for both methods was 0,98. The MIRU-VNTR test has the advantages of being simple, reproducible, non-subjective, which makes it suitable for large-scale screening of M. avium strains. Conclusions Both typing methods demonstrated a high degree of similarity between the strains of human and porcine origin. The parallel application of the methods adds epidemiological value to the comparison of the strains and their origins. The present approach and results support the hypothesis that there is a common source of M. avium subsp. hominissuis infection for pigs and humans or alternatively one species may be the infective source to the other.

In this study, a genotypification of Leishmania was performed using polimerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP) and sequencing techniques to identify species of Leishmania parasites in phlebotomine sand flies and dogs naturally infected. Between January-February of 2009, CDC light traps were used to collect insect samples from 13 capture sites in the municipality of Posadas, which is located in the province of Misiones of Argentina. Sand flies identified as Lutzomyia longipalpis were grouped into 28 separate pools for molecular biological analysis. Canine samples were taken from lymph node aspirates of two symptomatic stray animals that had been positively diagnosed with canine visceral leishmaniasis. One vector pool of 10 sand flies (1 out of the 28 pools tested) and both of the canine samples tested positively for Leishmania infantum by PCR and RFLP analysis. PCR products were confirmed by sequencing and showed a maximum identity with L. infantum. Given that infection was detected in one out of the 28 pools and that at least one infected insect was infected, it was possible to infer an infection rate at least of 0.47% for Lu. longipalpis among the analyzed samples. These results contribute to incriminate Lu. longipalpis as the vector of L. infantum in the municipality of Posadas, where cases of the disease in humans and dogs have been reported since 2005.

in 242 HIV-infected patients. Typing was based on DNA polymorphism of the ribosomal DNA ITS region of E. bieneusi. PCRRFLP generated with two restriction enzymes (Nla III and Fnu 4HI in PCR-amplified ITS products for classifying strains into different lineages. The diagnosis performance of the indirect immune-fluorescence-monoclonal antibody (IFI-AcM was defined in comparison with real-time PCR as the gold standard. Results. Out of 242 HIV-infected patients, using the real-time PCR, the prevalence of E. bieneusi was 7.9% (n=19 among the 19 E. bieneusi, one was coinfected with E. intestinalis. In 19 E. bieneusi persons using PCR-RFLP method, 5 type I strains of E. bieneusi (26.3% and 5 type IV strains of E. bieneusi (26.3% were identified. The sensitivity of IFI-AcM was poor as estimated 42.1%. Conclusion. Despite different PCR methods, there is possible association between HIVinfection, geographic location (France, Cameroun, Democratic Republic of Congo, and the concurrence of type I and type IV strains.

Objective. To determine the prevalence and the genotypes of Enterocytozoon bieneusi in stool specimens from HIV patients. Methods. This cross-sectional study was carried out in Kinshasa hospitals between 2009 and 2012. Detection of microsporidia including E. bieneusi and E. intestinalis was performed in 242 HIV-infected patients. Typing was based on DNA polymorphism of the ribosomal DNA ITS region of E. bieneusi. PCRRFLP generated with two restriction enzymes (Nla III and Fnu 4HI) in PCR-amplified ITS products for classifying strains into different lineages. The diagnosis performance of the indirect immune-fluorescence-monoclonal antibody (IFI-AcM) was defined in comparison with real-time PCR as the gold standard. Results. Out of 242 HIV-infected patients, using the real-time PCR, the prevalence of E. bieneusi was 7.9% (n = 19) among the 19 E. bieneusi, one was coinfected with E. intestinalis. In 19 E. bieneusi persons using PCR-RFLP method, 5 type I strains of E. bieneusi (26.3%) and 5 type IV strains of E. bieneusi (26.3%) were identified. The sensitivity of IFI-AcM was poor as estimated 42.1%. Conclusion. Despite different PCR methods, there is possible association between HIVinfection, geographic location (France, Cameroun, Democratic Republic of Congo), and the concurrence of type I and type IV strains. PMID:22811884

Full Text Available Abstract Humic substances in soil DNA samples can influence the assessment of microbial diversity and community composition. Using multiple steps during or after cell lysis adds expenses, is time-consuming, and causes DNA loss. A pretreatment of soil samples and a single step DNA extraction may improve experimental results. In order to optimize a protocol for obtaining high purity DNA from soil microbiota, five prewashing agents were compared in terms of their efficiency and effectiveness in removing soil contaminants. Residual contaminants were precipitated by adding 0.6 mL of 0.5 M CaCl2. Four cell lysis methods were applied to test their compatibility with the pretreatment (prewashing + Ca2+ flocculation and to ultimately identify the optimal cell lysis method for analyzing fungal communities in forest soils. The results showed that pretreatment with TNP + Triton X-100 + skim milk (100 mM Tris, 100 mM Na4P2O7, 1% polyvinylpyrrolidone, 100 mM NaCl, 0.05% Triton X-100, 4% skim milk, pH 10.0 removed most soil humic contaminants. When the pretreatment was combined with Ca2+ flocculation, the purity of all soil DNA samples was further improved. DNA samples obtained by the fast glass bead-beating method (MethodFGB had the highest purity. The resulting DNA was successfully used, without further purification steps, as a template for polymerase chain reaction targeting fungal internal transcribed spacer regions. The results obtained by terminal restriction fragment lengthpolymorphism analysis indicated that the MethodFGB revealed greater fungal diversity and more distinctive community structure compared with the other methods tested. Our study provides a protocol for fungal cell lysis in soil, which is fast, convenient, and effective for analyzing fungal communities in forest soils.

Full Text Available Abstract Background An observational case series was used to study the virulence characteristics and genotypes of paired Staphylococcus epidermidis isolates cultured from intraocular samples and from periocular environment of patients with postcataract surgery endophthalmitis. Methods Eight S. epidermidis isolates were obtained from three patients (2 from patients #1 and 2 and 4 from patient #3 whose vitreous and/or anterior chamber (AC specimens and preoperative lid/conjunctiva samples were culture positive. Cultures were identified by API-Staph phenotypic identification system and genotypically characterized by Fluorescent Amplified Fragment LengthPolymorphism (FAFLP and checked for their antimicrobial susceptibility. The isolates were tested for biofilm-production and methicillin-resistance (MR by PCR amplification of icaAB and mecA gene respectively. Results Four out of eight S. epidermidis strains showed multiple drug resistance (MDR. All the eight strains were PCR positive for mecA gene whereas seven out of eight strains were positive for icaAB genes. In all three patients FAFLP typing established vitreous isolates of S. epidermidis strains to be indistinguishable from the strains isolated from the patient's conjunctival swabs. However, from patient number three there was one isolate (1030b from lid swab, which appeared to be nonpathogenic and ancestral having minor but significant differences from other three strains from the same patient. This strain also lacked icaAB gene. In silico analysis indicated possible evolution of other strains from this strain in the patient. Conclusion Methicillin-resistant biofilm positive S. epidermidis strains colonizing the conjunctiva and eyelid were responsible for postoperative endophthalmitis (POE.

A better understanding of virus resistance mechanisms can offer more effective strategies to control virus diseases. Papaya ringspot virus (PRSV), Potyviridae, causes severe economical losses in papaya and cucurbit production worldwide. However, no resistance gene against PRSV has been identified to date. This study aimed to identify candidate PRSV resistance genes using cDNA-AFLP analysis and offered an open architecture and transcriptomic method to study those transcripts differentially expressed after virus inoculation. The whole genome expression profile of Cucumis metuliferus inoculated with PRSV was generated using cDNA-amplified fragment lengthpolymorphism (cDNA-AFLP) method. Transcript derived fragments (TDFs) identified from the resistant line PI 292190 may represent genes involved in the mechanism of PRSV resistance. C. metuliferus susceptible Acc. 2459 and resistant PI 292190 lines were inoculated with PRSV and subsequently total RNA was isolated for cDNA-AFLP analysis. More than 400 TDFs were expressed specifically in resistant line PI 292190. A total of 116 TDFs were cloned and their expression patterns and putative functions in the PRSV-resistance mechanism were further characterized. Subsequently, 28 out of 116 candidates which showed two-fold higher expression levels in resistant PI 292190 than those in susceptible Acc. 2459 after virus inoculation were selected from the reverse northern blot and bioinformatic analysis. Furthermore, the time point expression profiles of these candidates by northern blot analysis suggested that they might play roles in resistance against PRSV and could potentially provide valuable information for controlling PRSV disease in the future. PMID:23874746

Diagnosis and typing of Mycobacterium genus provides basic tools for investigating the epidemiology and pathogenesis of this group of bacteria. Polymerase chain reaction (PCR)-restriction fragment lengthpolymorphism analysis (PRA) is an accurate method providing diagnosis and typing of species of mycobacteria. The present study is conducted by the purpose of determining restriction fragment profiles of common types of mycobacteria by PRA method of rpoB gene in this geographical region. Totally 60 clinical and environmental isolates from February to October, 2013 were collected and subcultured and identified by phenotypic methods. A 360 bp fragment of the rpoB gene amplified by PCR and products were digested by MspI and HaeIII enzymes. In the present study, of all mycobacteria isolates identified by PRA method, 13 isolates (21.66%) were Mycobacterium tuberculosis, 34 isolates (56.66%) were rapidly growing Nontuberculosis Mycobacteria (NTM) that including 26 clinical isolates (43.33%) and 8 environmental isolates (13.33%), 11 isolates (18.33%) were clinical slowly growing NTM. among the clinical NTM isolates, Mycobacterium fortuitum Type I with the frequency of 57.77% was the most prevalent type isolates. Furthermore, an unrecorded of the PRA pattern of Mycobacterium conceptionense (HeaIII: 120/90/80, MspI: 120/105/80) was found. This study demonstrated that the PRA method was high discriminatory power for identification and typing of mycobacteria species and was able to identify 96.6% of all isolates. Based on the result of this study, rpoB gene could be a potentially useful tool for identification and investigation of molecular epidemiology of mycobacterial species.

The W-Beijing family is a widespread Mycobacterium tuberculosis clonal lineage that frequently causes epidemic outbreaks. This family is genetically homogeneous and conserved, so ETR-VNTR (exact tandem repeat-variable number of tandem repeats) typing is insufficient for strain differentiation, due to a common ETR-A to E profile (42435). This leads to the false clustering in molecular epidemiological studies, especially in the regions of predominance of the W-Beijing family. In this study, we searched for VNTR loci with a high evolutionary rate of polymorphism in the W-Beijing genome. Here we further evaluated VNTR typing on a set of 99 Mycobacterium tuberculosis clinical isolates and reference strains. These isolates were characterized and classified into several genotype families based on three ETR loci (A, C, E) and eight additional loci [previously described as QUB (Queen's University Belfast) or MIRU (Mycobacterial Interspersed Repetitive Units) or Mtubs]. Ninety-nine strains were divided into 74 VNTR-types, 51 isolates of the W-Beijing family identified by IS6110 RFLP-typing (the restriction fragment lengthpolymorphism-typing) and/or spoligotyping were subdivided into 30 VNTR-types. HGDI (the Hunter-Gaston discriminatory index) for all studied loci was close to that of IS6110 RFLP typing, a 'gold standard' method for subtyping M. tuberculosis complex strains. The QUB 26 and QUB 18 loci located in the PPE genes were highly polymorphic and more discriminative than other loci (HGDI is 0.8). Statistically significant increase of tandem repeats number in loci ETR-A, -E, QUB 26, QUB 18, QUB 11B, Mtub21 was revealed in the W-Beijing group compared to genetically divergent non-W-Beijing strains. Thirty-six isolates were subjected to IS6110 RFLP typing. The congruence between results of the IS6110 RFLP typing and 11-loci VNTR typing was estimated on 23 isolates of the W-Beijing family. These isolates were subdivided into 9 IS6110-RFLP types and 13 VNTR types. The poor

Renal diseases in cattle are frequently not recognized due to the subclinical conditions. Some species of Leptospira are the main cause of infectious agents that damage the kidneys and lead to abortion and economic losses in cattle and are also of major concern in the public health. This study was aimed to assess the prevalence of renal lesions of slaughtered cattle in the Shiraz abattoir and to determine the correlation between rejected kidneys and infection with Leptospira using nested PCR-restriction fragment lengthpolymorphism (RFLP) techniques. Out of 1000 inspected animals, 205 (20.5 %) revealed the renal lesions. Chronic nephritis (7.5 %), white-spotted kidney (7.3 %), and petechial hemorrhage (3.5 %) were the most prevalent forms of the lesions. A direct correlation between increasing the age and significant increase in the rate of lesions was also observed (P = 0.03). Using nested PCR-RFLP assay, 40.8 % of the tested kidneys were turned to be infected to the pathogenic species of Leptospira. The risk of infection of the kidneys with white spot to pathogenic species of Leptospira (53.8 %) was more than that of the kidneys with other lesions (25.0 %) (P = 0.014). The odd ratio indicates that the kidneys with white spot lesions are likely to be infected with pathogenic species of Leptospira, five times greater than other lesions. This study showed that renal lesions especially white-spotted kidney, which were considerably associated with Leptospira in slaughtered cattle in Shiraz, were very high. This is important in terms of public health and in particular, increases the risk of transmission of disease to human specially in the high-risk careers including farmers, veterinarians, and abattoir workers.

The composition of the intestinal microbiota was measured following consumption of identical meals for 3 days in 92 Japanese men, and terminal restriction fragment lengthpolymorphism (T-RFLP) was used to analyze their feces. The obtained operational taxonomic units (OTUs) and the subjects' ages were classified by using Data mining (DM) software that compared these data with continuous data and for 5 partitions for age divided at 5 years intervals between the ages of 30 and 50. The DM provided Decision trees in which the selected OTUs were closely related to the ages of the subjects. DM was also used to compare the OTUs from the T-RFLP data with seven restriction enzymes (two enzymes of 516f-BslI and 516f-HaeIII, two enzymes of 27f-MspI and 27f-AluI, three enzymes of 35f-HhaI, 35f-MspI and 35f-AluI) and their various combinations. The OTUs delivered from the five enzyme-digested partitions were analyzed to classify their age clusters. For use in future DM processing, we discussed the enzymes that were effective for accurate classification. We selected two OTUs (HA624 and HA995) that were useful for classifying the subject's ages. Depending on the 16S rRNA sequences of the OTUs, Ruminicoccus obeum clones 1-4 were present in 18 of 36 bacterial candidates in the older age group-related OTU (HA624). On the other hand, Ruminicoccus obeum clones 1-33 were present in 65 of 269 candidates in the younger age group-related OUT (HA995).

Methylenetetrahydrofolate reductase plays a central role in converting folate to methyl donor for DNA methylation. Recently, methylenetetrahydrofolate reductase (MTHFR C677T and A1298C) mutations were discovered to be associated with childhood acute lymphoblastic leukemia (ALL), as well as colon cancer, lymphoma, esophageal and stomach cancer. Therefore, it was hypothesized that the MTHFR polymorphisms are associated with the risk of childhood ALL in the Korean population. DNA samples taken from 66 patients with ALL and 100 age-matched controls were analyzed using the polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP) assay for detection of MTHFR C677T and A1298C mutations. The frequency of the AC genotype for MTHFR A1298C polymorphism was significantly different between the controls and the cases (OR, 2.22; CI, 95% 1.09-4.51, p=0.03). The 1298AC+CC genotype was also significantly different (OR, 2.11; 95% CI, 1.06-4.22; p=0.049). There was, however, no significant difference for MTHFR C677T polymorphism and combined genotype frequencies between the two groups. Although no consistent results on associations between MTHFR A 1298C polymorphism and ALL in the populations studied were obtained, the A1298C polymorphism, at least in Koreans, may be a genetic determinant among childhood ALL patients.

The vitamin D receptor (VDR) gene serves as a good candidate gene for susceptibility to several diseases. The gene has a critical role in regulating the renin-angiotensin system (RAS) influencing the regulation of blood pressure. Hence determining the association of VDR polymorphisms with essential hypertension is expected to help in the evaluation of risk for the condition. The aim of this study was to evaluate association between VDRFok I polymorphism and genetic susceptibility to essential hypertension. Two hundred and eighty clinically diagnosed hypertensive patients and 200 normotensive healthy controls were analyzed for Fok I (T/C) [rs2228570] polymorphism by polymerase chain reaction and restriction fragment lengthpolymorphism (PCR-RFLP) analysis. Genotype distribution and allele frequencies in patients and controls, and odds ratios (ORs) were calculated to predict the risk for developing hypertension by the individuals of different genotypes. The genotype distribution and allele frequencies of Fok I (T/C) [rs2228570] VDR polymorphism differed significantly between patients and controls (χ(2) of 18.0; 2 degrees of freedom; P = 0.000). FF genotype and allele F were at significantly greater risk for developing hypertension and the risk was elevated for both the sexes, cases with positive family history and habit of smoking. Our data suggest that VDR gene Fok I polymorphism is associated with the risk of developing essential hypertension.

Full Text Available Oxidative stress is implicated in the pathogenesis of many diseases, including serious ocular diseases, keratoconus (KC and Fuchs endothelial corneal dystrophy (FECD. Flap endonuclease 1 (FEN1 plays an important role in the repair of oxidative DNA damage in the base excision repair pathway. We determined the association between two single nucleotide polymorphisms (SNPs, c.–441G>A (rs174538 and g.61564299G>T (rs4246215, in the FEN1 gene and the occurrence of KC and FECD. This study involved 279 patients with KC, 225 patients with FECD and 322 control individuals. Polymerase chain reaction (PCR and lengthpolymorphism restriction fragment analysis (RFLP were applied. The T/T genotype of the g.61564299G>T polymorphism was associated with an increased occurrence of KC and FECD. There was no association between the c.–441G>A polymorphism and either disease. However, the GG haplotype of both polymorphisms was observed more frequently and the GT haplotype less frequently in the KC group than the control. The AG haplotype was associated with increased FECD occurrence. Our findings suggest that the g.61564299G>T and c.–441G>A polymorphisms in the FEN1 gene may modulate the risk of keratoconus and Fuchs endothelial corneal dystrophy.

Background: Recurrent pregnancy loss (RPL) is a multifactorial disorder. Environmental factors and genetics can affect pregnancy outcomes. Objective: Conflicting data suggest an association between estrogen receptor alpha (ESR1) gene polymorphisms and RPL. In this study, such association was investigated in Iranian women with RPL. Materials and Methods: In this case control study, blood samples were collected from 244 women with a history of three or more consecutive pregnancy losses and 104 healthy women with at least two live births. Using polymerase chain reaction- restriction fragment lengthpolymorphism (PCR-RFLP), we studied -397C/T and -351A/G polymorphisms on ESR1 gene in case and control subjects. Results: The genotypic frequencies of -397C/T and -351A/G polymorphisms on ESR1were not significantly different between RPL and control groups (p=0.20 and p=0.09, respectively). A significantly negative correlation was observed between -397C/T and -351A/G (r=-0.852, p<0.001) in RPL women and complete linkage disequilibrium between the investigated polymorphisms was found (D’: 0.959; r-square= 0.758, p<0.001). Conclusion: This investigation suggests that the analyzed polymorphisms on ESR1gene are not associated with an increased risk of RPL in the studied population. PMID:25071847

Mutations in the mitochondrial DNA (mtDNA) have been reported in a wide variety of human neoplasms. A polynucleotide tract extending from 303 to 315 nucleotide positions (D310) within the non-coding region of mtDNA has been identified as a mutational hotspot of primary tumors. This region consists of two polycytosine stretches interrupted by a thymidine nucleotide. The number of cytosines at the first and second stretches are 7 and 5 respectively, according to the GeneBank sequence. The first stretch exhibits a polymorphiclength variation (6-C to 9-C) among individuals and has been investigated in many cancer types. Large-scale studies are needed to clarify the relationship between cytosine number and cancer development/progression. However, time and money consuming methods such as radioactivity-based gel electrophoresis and sequencing, are not appropriate for the determination of this polymorphism for large case-control studies. In this study, we conducted a rapid RFLP analysis using a restriction enzyme, BsaXI, for the single step simple determination of 7-C carriers at the first stretch in D310 region. 25 colorectal cancer patients, 25 breast cancer patients and 41 healthy individuals were enrolled into the study. PCR amplification followed by restriction enzyme digestion of D310 region was performed for RFLP analysis. Digestion products were analysed by agarose gel electrophoresis. Sequencing was also applied to samples in order to confirm the RFLP data. Samples containing 7-C at first stretch of D310 region were successfully determined by the BsaXI RFLP method. Heteroplasmy and homoplasmy for 7-C content was also determined as evidenced by direct sequencing. Forty-one percent of the studied samples were found to be BsaXI positive. Furthermore, BsaXI status of colorectal cancer samples were significantly different from that of healthy individuals. In conclusion, BsaXI RFLP analysis is a simple and rapid approach for the single step determination of D310

Full Text Available Simple double repetitive element polymerase chain reaction (MaDRE-PCR and Pvu II-IS1245 restriction fragment lengthpolymorphism (RFLP typing methods were used to type 41 Mycobacterium avium isolates obtained from 14 Aids inpatients and 10 environment and animals specimens identified among 53 mycobacteria isolated from 237 food, chicken, and pig. All environmental and animals strains showed orphan patterns by both methods. By MaDRE-PCR four patients, with multiple isolates, showed different patterns, suggesting polyclonal infection that was confirmed by RFLP in two of them. This first evaluation of MaDRE-PCR on Brazilian M. avium strains demonstrated that the method seems to be useful as simple and less expensive typing method for screening genetic diversity in M. avium strains on selected epidemiological studies, although with limitation on analysis identical patterns except for one band.

Full Text Available Carpal tunnel syndrome (CTS is the most common entrapment neuropathy of the upper extremity. In this study, we aimed to clarify the relationships between the catechol-O-methyltransferase (COMT gene Val158Met (rs4680 polymorphism and development, functional and clinical status of CTS. Ninety-five women with electro diagnostically confirmed CTS and 95 healthy controls were enrolled in the study. The functional and clinical status of the patients was measured by the Turkish version of the Boston Questionnaire and intensity of pain related to the past 2 weeks was evaluated on a visual analog scale (VAS. The Val158Met polymorphism was determined using the polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP, method. We divided patients according to the genotypes of the Val158Met polymorphism as Val/Val, Val/Met and Met/Met. There were not any significant differences in terms of Val158Met polymorphisms between patients and healthy controls (p >0.05. We also did not find any relationships between the Val158Met polymorphism and CTS (p >0.05. In conclusion, although we did not find any relationships between CTS and the Val158Met polymorphism, we could not generalize this result to the general population. Future studies are warranted to conclude precise associations.

Full Text Available Abstract Background Hepatocellular carcinoma (HCC is one of the most frequent malignant neoplasms in the world. Genetic polymorphism has been reported to be a factor increasing the risk of HCC. Phase II enzymes such as glutathione s-transferases (GSTP1, GSTA1 play important roles in protecting cells against damage induced by carcinogens. The aim of this study was to estimate the relationship of the GSTP1 and GSTA1 gene polymorphisms to HCC risk and clinico-pathological status. Methods Polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP was used to measure GSTP1 (A→G and GSTA1 (C→T gene polymorphisms in 386 healthy controls and 177 patients with HCC. Results Neither gene polymorphism was associated with the clinico-pathological status of HCC and serum expression of liver-related clinico-pathological markers. No association between the GSTA1 gene polymorphism and HCC susceptibility was found. However, in the younger group, aged ≤ 57 years, individuals with AG or GG alleles of GSTP1 had a 2.18-fold (95%CI = 1.09-4.36; p = 0.02 and 5.64-fold (95%CI = 1.02-31.18; p = 0.04 risk, respectively, of developing HCC compared to individuals with AA alleles, after adjusting for other confounders. Conclusion AG and GG alleles of GSTP1 gene polymorphisms may be considered as factors increasing the susceptibility to and risk of HCC in Taiwanese aged ≤ 57 years.

To study the correlation of G870A CCND1 gene polymorphism and digestive system tumors. From August 2010 to August 2014, 164 digestive system cancer patients (including 82 patients with gastric cancer and 82 with colorectal cancer) and 82 healthy subjects (control group) were examined with PCR-restriction fragment lengthpolymorphism (PCR-RFLP). The distribution of CCND1 gene G870A frequency in the 3 groups and its association with tumor staging and grading were analyzed. The frequencies of the GG, GA and AA genotypes in G870A CCND1 gene loci in patients with gastric cancer and colorectal cancer differed significantly from those in the control group (Pdigestive system tumors (Pdigestive system cancer risk than the GG genotype (Pdigestive system tumors. The allele A is associated with an increased risk of digestive system tumors and correlated with the tumor differentiation and staging of the tumor.

Full Text Available Huge efforts have been invested in the last two decades to dissect the genetic bases of complex traits including yields of many crop plants, through quantitative trait locus (QTL analyses. However, almost all the studies were based on linkage maps constructed using low-throughput molecular markers, e.g. restriction fragment lengthpolymorphisms (RFLPs and simple sequence repeats (SSRs, thus are mostly of low density and not able to provide precise and complete information about the numbers and locations of the genes or QTLs controlling the traits. In this study, we constructed an ultra-high density genetic map based on high quality single nucleotide polymorphisms (SNPs from low-coverage sequences of a recombinant inbred line (RIL population of rice, generated using new sequencing technology. The quality of the map was assessed by validating the positions of several cloned genes including GS3 and GW5/qSW5, two major QTLs for grain length and grain width respectively, and OsC1, a qualitative trait locus for pigmentation. In all the cases the loci could be precisely resolved to the bins where the genes are located, indicating high quality and accuracy of the map. The SNP map was used to perform QTL analysis for yield and three yield-component traits, number of tillers per plant, number of grains per panicle and grain weight, using data from field trials conducted over years, in comparison to QTL mapping based on RFLPs/SSRs. The SNP map detected more QTLs especially for grain weight, with precise map locations, demonstrating advantages in detecting power and resolution relative to the RFLP/SSR map. Thus this study provided an example for ultra-high density map construction using sequencing technology. Moreover, the results obtained are helpful for understanding the genetic bases of the yield traits and for fine mapping and cloning of QTLs.

Full Text Available In Cuba, several Biomphalaria species have been reported such as B. orbignyi, B. schrammi, B. helophila, B. havanensis and B. peregrina; only the latter three are considered as potential hosts of Schistosoma mansoni. The specific identification of Biomphalaria species is based on anatomical and morphological characters of genital organs and shells. The correct identification of these snails is complicated by the high variation in these characters, similarity among species and in some cases by the small size of the snails. In this paper, we reported the classical morphological identification, the use of PCR and RFLP analysis of the internal transcribed spacer region of the ribosomal RNA genes for molecular identification of seven snail populations from different localities in Cuba. Using morphological and molecular analysis, we showed that among the studied Cuban Biomphalaria populations only B. havanensis and B. obstructa species were found.

Full Text Available Diagnosis of the Mycobacterium tuberculosis complex by direct PCR of mediastinal lymphnode DNA and microbiological tests were compared in cattle suspicious of bearing tuberculous-like lesions detected during slaughter. The PCR procedure applied on DNA samples (n=54 obtained by adding alpha -casein into the thiocyanate extraction mix was positive in 70% of the samples. PCR confirmed the identification of 23 samples (100% that grew in culture, 9 samples (60% that failed to grow in culture, plus 6 (37.5% samples that resulted in growth of bacterial contaminants. Genotyping by IS6110-RFLP and DR-spoligotyping analysis of seven samples revealed the presence of several polimorphisms. Seven of the isolates contained multiple copies of IS6110, thus defining the existence of five singular genotypes.

The most important traits of Chinese Liaoning cashmere goat fiber are fiber diameter, weight, and length. We looked for polymorphisms and their possible association with cashmere fiber traits in the 5' upstream region (5' UTR) of the prolactin receptor gene (PRLR), which encodes an anterior pituitary peptide hormone involved in different physiological activities; it is the principal endocrine regulator in pelage replacement in mammals. A novel single-nucleotide polymorphism (SNP) was found in the 5' UTR of PRLR by PCR-RFLP in an analysis of 590 goats. Two genotypes (CC and CT) were observed. The frequencies of allele C and T were 0.93 and 0.07, respectively. Association analysis revealed that the PRLR 5' UTR polymorphism (SNP5) was significantly associated with cashmere fiber weight and diameter. This novel SNP in hircine PRLR has potential as a molecular marker for cashmere fiber weight and diameter in Liaoning cashmere goats.

Full Text Available Inter- and intraspecific variation among 26 isolates of ectomycorrhizal fungi belonging to 8 genera and 19 species were evaluated by analysis of the internal transcribed sequence (ITS of the rDNA region using restriction fragment lengthpolymorphism (RFLP. The ITS region was first amplified by polymerase chain reaction (PCR with specific primers and then cleaved with different restriction enzymes. Amplification products, which ranged between 560 and 750 base pairs (bp, were obtained for all the isolates analyzed. The degree of polymorphism observed did not allow proper identification of most of the isolates. Cleavage of amplified fragments with the restriction enzymes Alu I, Hae III, Hinf I, and Hpa II revealed extensive polymorphism. All eight genera and most species presented specific restriction patterns. Species not identifiable by a specific pattern belonged to two genera: Rhizopogon (R. nigrescens, R. reaii, R. roseolus, R. rubescens and Rhizopogon sp., and Laccaria (L. bicolor and L. amethystea. Our data confirm the potential of ITS region PCR-RFLP for the molecular characterization of ectomycorrhizal fungi and their identification and monitoring in artificial inoculation programs.

Full Text Available Triatomines are insects of great importance for public health because they transmit the parasite Trypano-soma cruzi, etiological agent of Chagas disease. These insects harbour intestinal symbiotic flora from theRhodococcus genus, which provide the nutrients lacking in the insect diet, nutrients which are essentialfor normal insect development and growth. Study of these bacteria is of special interest due to thepossibility of designing paratransgenesis control strategies as well as for implications that interactionsbetween these bacteria and parasites can develop inside triatomine insects. Taking into account thedifficulty in identifying bacteria from the Rhodococcus genus by traditional methods, in this work apolymerase chain reaction (PCR followed by a restriction fragment lengthpolymorphism analysis wasstandardized to identify Rhodococcus rhodnii isolated from Rhodnius prolixus. Gene coding for bacterial16 S rRNA was amplified and obtained products were sequenced and digested with several restrictionenzymes. Results show a total correlation between number and size of the restriction fragments obtainedin vitro and in silico as well. Therefore, applicability of this PCR-RFLP assay for R. rhodnii identificationhas been demonstrated.

Full Text Available The gram negative facultative bacterium P. salmonis is the etiological agent of Salmonid Rickettsial Septicaemia (SRS, a severe disease that causes important economic losses in the global salmon farmer industry. Despite efforts to control this disease, the high frequency of new epizootic events indicate that the vaccine and antibiotics treatments have limited effectiveness, therefore the preventive and diagnostic approaches must be improved. A comparison of several methodologies for SRS diagnostic indicate differences in their specificity and its capacity to detect other bacteria coexisting with P. salmonis in culture media (contamination and fish samples (coinfection, aspects relevant for research, vaccine development and clinical diagnostic. By computer-simulation analyses, we identified a group of restriction enzymes that generate unique P. salmonis 16S rDNA band patterns, distinguishable from all other bacteria. From this information, we designed and developed a PCR-RFLP (Polymerase Chain Reaction - Restriction Fragment LengthPolymorphism assay, which was validated using 16S rDNA universal primers and restriction enzyme PmaCI for the amplification and digestion, respectively. Experimental validation was performed by comparing the restriction pattern of P. salmonis with the restriction patterns generated by bacteria that cohabit with P. salmonis (fish bacterial isolates and culture media contaminants. Our results indicate that the restriction enzyme selection pipeline was suitable to design a more specific, sensible, faster and cheaper assay than the currently used P. salmonis detection methodologies.

Chronic obstructive pulmonary disease (COPD) is a progressive, inflammatory lung disease that affects a large number of patients and has significant impact. One hallmark of the disease is the presence of bacteria in the lower airways. The aim of this study was to analyze the detailed structure of microbial communities found in the lungs of healthy individuals and patients with COPD. Nine COPD patients as compared and 9 healthy individuals underwent flexible bronchoscopy and BAL was performed. Bacterial nucleic acids were subjected to terminal restriction fragment (TRF) lengthpolymorphism and clone library analysis. Overall, we identified 326 T-RFLP band, 159 in patients and 167 in healthy controls. The results of the TRF analysis correlated partly with the data obtained from clone sequencing. Although the results of the sequencing showed high diversity, the genera Prevotella, Sphingomonas, Pseudomonas, Acinetobacter, Fusobacterium, Megasphaera, Veillonella, Staphylococcus, and Streptococcus constituted the major part of the core microbiome found in both groups. A TRF band possibly representing Pseudomonas sp. monoinfection was associated with a reduction of the microbial diversity. Non-cultural methods reveal the complexity of the pulmonary microbiome in healthy individuals and in patients with COPD. Alterations of the microbiome in pulmonary diseases are correlated with disease.

Full Text Available Renin angiotensin system (RAS comprising Angiotensin converting enzyme (ACE, Angiotensin II (Ang II and its receptor Angiotensin II receptor type I (AGTR1, plays a critical role in several diseases including cancer. A single nucleotide polymorphism (SNP A1166C located in 3′ untranslated region (UTR of AGTR1 and an insertion/deletion (I/D polymorphism present in intron 16 of ACE gene have been associated with many diseases, but their association with Breast cancer (BCa is still debatable. Here, we for the first time investigated the association of these polymorphisms in a North Indian BCa cohort including 161 patients and 152 healthy women. The polymorphisms were evaluated by polymerase chain reaction (PCR and restriction fragment lengthpolymorphism (RFLP respectively. The association between these polymorphisms and BCa risk was estimated by calculating Odds Ratio (OR and chi-square (χ2 test. The DD genotype/D allele of ACE (I/D polymorphism and “AC and CC” genotype/C allele of AGTR1 (A1166C polymorphism were associated with higher risk of BCa when evaluated independently. Furthermore, interaction analysis of “AC and CC” and DD genotype and combination of “C and D” alleles of both polymorphisms revealed significantly greater BCa risk than that observed independently. Conclusively, women harboring “AC or CC” genotype/C allele for AGTR1 (A1166C polymorphism and DD genotype/D allele for ACE (I/D polymorphisms have a predisposition to develop more aggressive disease with advanced staging and larger tumor size. Our study indicates importance of genetic screening based on these polymorphisms for women, who may have higher risk of BCa.

Full Text Available The tyrosine kinase receptor RET, which is the protein product of the RET gene, is involved in the development of the mammalian nervous system that causes Hirschsprung’s disease (HSCR. RETs are cell surface molecules that are expressed in cells derived from the neural crest. The purpose of this study was to investigate the polymorphism of the RET gene in HSCR in the Yogyakarta population. Genomic DNA was extracted from surgically removed bowel tissues of 54 unrelated HSCR patients. Exon 2 of the RET gene was amplified by polymerase chain reaction (PCR and analyzed by restriction fragment lengthpolymorphism (RFLP. Molecular results were compared with clinical performance of Hirschsprung patients. RET polymorphism was detected in exon 2 in all of the 54 Indonesian HSCR patients. The allelic distribution of the c135GàA polymorphism in the RET exon 2 indicated that the A allele was more frequent in patients than in control individuals (chi-square test, p= 0.001. Thus the RET variant allele A is over-represented in patients affected with the HSCR phenotype. Polymorphism of exon 2 of the RET gene was found in sporadic Hirschsprung’s disease in the Yogyakarta population, which suggests that the RET gene plays important roles in the pathogenesis of HSCR.

Full Text Available The tyrosine kinase receptor RET, which is the protein product of the RET gene, is involved in the development of the mammalian nervous system that causes Hirschsprung’s disease (HSCR. RETs are cell surface molecules that are expressed in cells derived from the neural crest. The purpose of this study was to investigate the polymorphism of the RET gene in HSCR in the Yogyakarta population. Genomic DNA was extracted from surgically removed bowel tissues of 54 unrelated HSCR patients. Exon 2 of the RET gene was amplified by polymerase chain reaction (PCR and analyzed by restriction fragment lengthpolymorphism (RFLP. Molecular results were compared with clinical performance of Hirschsprung patients. RET polymorphism was detected in exon 2 in all of the 54 Indonesian HSCR patients. The allelic distribution of the c135GàA polymorphism in the RET exon 2 indicated that the A allele was more frequent in patients than in control individuals (chi-square test, p= 0.001. Thus the RET variant allele A is over-represented in patients affected with the HSCR phenotype. Polymorphism of exon 2 of the RET gene was found in sporadic Hirschsprung’s disease in the Yogyakarta population, which suggests that the RET gene plays important roles in the pathogenesis of HSCR.

Full Text Available The first signs of oral cancer may resemble developing infections in the mucous membranes, with throat cancer symptoms being similar to those of upper respiratory tract infections. This greatly hinders rapid diagnosis and treatment. Better knowledge of the changes occurring in the metabolism of folic acid can help in understanding the carcinogenesis affecting DNA methylation and genome stability. Polymorphisms in genes encoding enzymes involved in this pathway may influence enzyme activity and thereby interfere with the concentrations of homocysteine and S-adenosylmethionine, which are important for DNA synthesis and cellular methylation reactions. The aim of the study was to determine the risk of oral cancer associated with the TC2 C776G polymorphism, as determined in 119 patients. Genotypes were determined by polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP. The test genotype was found to correspond to the Hardy-Weinberg (HW equilibrium (p > 0.05. In our population G/G homozygosity of C776G TC2 gene polymorphism increases the risk of oral cancer; OR (odds ratio: 4.3875; 95% CI (confidence interval: 2.0518-9.319; p = 0.001. Regarding C/G genotype of the C776G TC2 gene, polymorphism also increases the risk of developing this cancer; OR 2.4146 95% CI: 1.2803-4.5541; p = 0.01.

Eosinophil cationic protein (ECP), a potent cytotoxic molecule, is released by activated eosinophils. ECP has been suggested to be involved in tissue remodeling of allergic diseases. The ECP (RNase3) gene is a candidate gene in atopic diseases. RNase3 polymorphisms have been reported to have an association with atopy. We determined whether polymorphisms in the RNase3 gene are associated with allergic rhinitis in a Korean population. The Taqman assay, restriction fragment lengthpolymorphism (PCR-RFLP), and high-resolution melt (HRM) were used for genotyping. Three single nucleotide polymorphisms (SNPs; g.-550A>G, g.371G>C, and g.499G>C) were identified. The genotype of the SNPs was analyzed in patients with allergic rhinitis and controls without allergic rhinitis. The genotype and allele frequencies were compared between both groups. The genotype frequencies of the g.-550A>G and g.371G>C SNPs were not significantly different between patients with allergic rhinitis and controls (P > 0.05). However, in patients with allergic rhinitis, the genotype and allele frequencies of the g.499G>C SNP of RNase 3 were significantly different from those of the control group (P associated with allergic rhinitis (P = 0.048), while the G-G-G haplotype was negatively associated with allergic rhinitis (P = 0.004). Our study suggests that RNase3 polymorphisms are potentially associated with susceptibility to allergic rhinitis.

Full Text Available The pathogenesis of essential hypertension (EH is affected by genetic and environmental factors. Mutations in hypertension-related genes can affect blood pressure (BP via alteration of salt and water reabsorption by the nephron. The genes of the renin-angiotensin system (RAS have been extensively studied because of the well documented role of this system in the control of BP. It has been previously shown that Angiotensin II type 1 receptor (ATR1 gene polymorphism could be associated with increased risk of EH. So, in the current study, we evaluated the frequency of ATR1 (A1166C polymorphism in relation to EH in a group of Egyptian population. The study population included 83 hypertensive patients and 60 age and sex matched healthy control subjects. Restriction fragment lengthpolymorphism – Polymerase chain reaction (RFLP – PCR was used for the analysis of A1166C polymorphism of ATR1 genes in peripheral blood samples of all patients and controls. The results revealed that there was a positive risk of developing EH when having the T allele whether in homozygous or heterozygous state. From this work, it was concluded that there was an association between ATR1 (A1166C gene polymorphism and the risk of developing EH.

Full Text Available The pathogenesis of essential hypertension (EH is affected by genetic and environmental factors. Mutations in hypertension-related genes can affect blood pressure (BP via alteration of salt and water reabsorption by the nephron. The genes of the renin-angiotensin system (RAS have been extensively studied because of the well documented role of this system in the control of BP. It has been previously shown that angiotensinogen (AGT gene polymorphism could be associated with increased risk of EH. The current study evaluated the frequency of AGT (M235T polymorphism in relation to EH in a group of Egyptian population. The study population included 83 hypertensive patients and 60 age and sex matched healthy control subjects. Restriction fragment lengthpolymorphism-Polymerase chain reaction (RFLP-PCR was used for the analysis of M235T polymorphism of AGT genes in peripheral blood samples of all patients and controls. The results revealed that there was a positive risk of developing EH when having the T allele whether in homozygous or heterozygous state. It was concluded that there was an association between AGT (M235T gene polymorphism and the risk of developing EH.

Stroke is the second leading cause of death in the world and in South Korea. Ischemic stroke and silent brain infarction (SBI) are complex, multifactorial diseases influenced by multiple genetic and environmental factors. Moderately elevated plasma homocysteine levels are a major risk factor for vascular diseases, including stroke and SBI. Folate and vitamin B12 are important regulators of homocysteine metabolism. Reduced folate carrier (RFC), a bidirectional anion exchanger, mediates folate delivery to a variety of cells. We selected three known RFC-1 polymorphisms (-43C>T, 80A>G, 696T>C) and investigated their relationship to cerebral infarction in the Korean population. We used the polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP) method to analyze associations between the three RFC-1 polymorphisms, disease status, and folate and homocysteine levels in 584 ischemic stroke patients, 353 SBI patients, and 505 control subjects. The frequencies of the RFC-1 -43TT, 80GG, and 696CC genotypes differed significantly between the stroke and control groups. The RFC-1 80A>G substitution was also associated with small artery occlusion and SBI. In a gene-environment analysis, the RFC-1 -43C>T, 80A>G, and 696T>C polymorphisms in the ischemic stroke group had combined effects with all environmental factors. In summary, we found that the RFC-1 -43C>T, 80A>G, and 696T>C polymorphisms may be risk factors for ischemic stroke.

The objectives of this study aimed to detect a CYP2B6 polymorphism in de novo cases of acute myeloid leukemia patients and identify any role in disease progression and outcome. DNA was isolated from peripheral blood of 82 newly diagnosed acute myeloid leukemia cases and the CYP2B6 G15631T gene polymorphism was assayed by PCR restriction fragment lengthpolymorphism (PCR-RFLP). The frequency of the GG genotype (wild type) was 48 (58.5%) and that of the mutant type T allele was 34 (41.9%). GT genotype heterozygous variants were found in 28 (34%), and TT genotype homozygous variants in 6 (7.3%) cases. We found no significant association between the CYP2B6 G15631T polymorphism and complete response (CR) (p-value=0.768), FAB classification (p-value=0.51), cytogenetic analysis (p-value=0.673), and overall survival (p-value=0.325). Also, there were no significant links with early toxic death (p-value=0.92) or progression- free survival (PFS) (p-value=0.245). Our results suggest that the CYP2B6 polymorphism has no role in disease progression, therapeutic outcome, patient free survival, early toxic death and overall survival in acute myeloid leukemia patients.

Full Text Available Migraine is a common neurovascular brain disorder with heterogeneous clinical presentation, including recurrent headache attacks. The pathophysiology of migraine is complex, and a number of genomic regions have been associated with the development of migraine. In this study, we analyzed the allele and genotype frequencies of the urotensin-II gene (UTS2 polymorphisms, Thr21Met and Ser89Asn, among Turkish patients with migraine. A total of 146 patients with migraine (14 with aura [MA group] and 132 without aura [MO group] were genotyped for Thr21Met and Ser89Asn polymorphisms and compared with 154 age- and sex-matched healthy controls. The UTS2 gene polymorphisms were analyzed by polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP. No significant differences were observed in allele and genotype frequencies for Thr21Met and Ser89Asn polymorphisms between the patients with migraine and control group. Similarly, we did not observe significant differences in allele and genotype frequencies between MA and MO and control group. Moreover, the haplotype analysis showed no association between UTS2 gene haplotypes (MN, MS, TN, and TS and migraine. In summary, Thr21Met and Ser89Asn polymorphisms of the UTS2 gene are not risk factors for migraine in our sample of Turkish migraine patients.

Full Text Available ABSTRACT INTRODUCTION: Oral squamous cell carcinoma (OSCC is a serious public health problem, due to its high mortality rate and worldwide rising incidence. OSCC susceptibility is mediated by interactions between genetic and environmental factors. Studies suggest that genetic variants encoding enzymes involved in folate metabolism may modulate OSCC risk by altering DNA synthesis/repair and methylation process. OBJECTIVE: The goals of this study were to evaluate the association of three genotypic polymorphism (MTHFR C677T, MTHFR A1298C and CBS 844ins68 and oral cancer risk in southeastern Brazilians and evaluate the interactions between polymorphisms and clinical histopathological parameters. METHODS: This case-control study included 101 cases and 102 controls in the state of Espírito Santo, Brazil. MTHFR genotyping was done by PCR-RFLP (polymerase chain reaction - restriction fragment lengthpolymorphism and CBS genotyping by PCR (polymerase chain reaction analysis. RESULTS: MTHFR C677T polymorphism was associated with lymph node involvement. Genotype CT + TT acted as a protective factor. MTHFR A1298C AC + CC genotype was associated with tumor differentiation, and possibly with a better prognosis. In risk analysis, no correlation was observed between genotypes and OSCC. CONCLUSION: We concluded that MTHFR C677T, MTHFR A1298C and CBS 844ins68 polymorphisms were not associated with OSCC risk in southeastern Brazilians; however, we suggest a prognosis effect associated with MTHFR C677T and A1298C polymorphisms in OSCC.

Full Text Available Interleukin I (lL-I is selectively cytotoxic to the insulin producing beta cell of pancreatic islets. This effect may be due to IL-I induced generation of reactive oxygen species and nitric oxide. Since beta cells contain low amounts of the superoxide radical scavenger enzyme manganese superoxide dismutase (MnSOD, this may leave beta cells more susceptible to IL-I than other cell types. Genetic variation in the MnSOD locus could reflect differences in scavenger potential. We, therefore, studied possible restriction fragment lengthpolymorphisms (RFLPs of this locus in patients with insulin-dependent diabetes mellitus (100M (n= 154 and control individuals (n=178, Taql revealed a double diallelic RFLP in patients as well as in controls. No overall difference in allelic or genotype frequencies were observed between 100M patients and control individuals (p=0.11 and no significant association of any particular RFLP pattern with 100M was found. Structurally polymorphic MnSOD protein variants with altered activities have been reported. If genetic variation results in MnSOD variants with reduced activities, the MnSOD locus may still be a candidate gene for 100M susceptibility. Whether the RFLPs reported in this study reflects differences in gene expression level, protein level and/or specific activity of the protein is yet to be studied.

Full Text Available ABSTRACT Introduction: Cancer is considered a genetic disease. For this reason, identification and characterization of the genes involved in its origin and progression are of fundamental importance in understanding its molecular basis. Objective: Our objective was to determine whether people from Macapá with a diagnosis of cancer have genetic polymorphisms related to the XRCC1 gene. Materials and methods: We analyzed 30 samples of deoxyribonucleic acid (DNA of cases with cancer and 30 control samples. All samples were amplified and analyzed by the polymerase chain reaction-restriction fragment lengthpolymorphisms (PCR-RFLP method, with the use of restriction enzyme MspI. Results: Regarding the 194T polymorphism, we found that all samples of the cases presented the polymorphic allele Trp (Arg/Trp. In control samples, 96.6% also identify the polymorphic allele Trp and, among these, one was homozygous for the same allele (Trp/Trp. Regarding the 399A polymorphism, 83.3% of the cases and 23.3% of the controls had the Arg/ Gln genotype, respectively. We found that 73.3% of controls and 16.6% of cases had the Arg/Arg genotype. Among the controls, we found only a sample that was homozygous for the polymorphic allele Trp/Trp. Conclusion: Our results demonstrated the allele frequency of 194Trp polymorphism in both sample groups analyzed. We also found a significant number of polymorphic allele 399A in people with cancer. Thus, we can highlight 399Gln polymorphism as a genetic marker of cancer risk in this population.

B-G antigens are cell-surface molecules encoded by a highly polymorphic multigene family located in the chicken major histocompatibility complex (MHC). Rabbit antisera to B-G molecules immunoprecipitate 3-6 bands from iodinated erythrocytes by sodium dodecyl sulfate (SDS) gels under reducing...... conditions. These are all B-G molecules because they all map to the B-G region of the chicken MHC in congenic and recombinant chickens, most are directly recognized by the antisera, most form disulfide-linked dimers, and none bear N-linked carbohydrate. Both apparent homodimers and heterodimers are found......, which bear intrachain disulfide bonds. All 3-6 bands have different mobilities in SDS gels between different haplotypes, ranging from 30 to 55 kDa. This size polymorphism is not affected by glycosidase treatment or addition of protease inhibitors. Partial proteolysis of cell surface-iodinated B-G...

Pituitary Homeobox 2 (PITX2), is an active gene as a paired-related homeobox gene that encodes multiple isoforms. Its Nodal pathway in determination of left-right patterning during embryogenesis has been reported in satellite cells and expressed in adult human skeletal muscle. PITX2A and PITX2B are produced by alternative splicing and used of different promoters. PITX2C uses an alternative promoter located upstream of exon 4. PITX2D is produced by PITX2C alternative promoter and differential splicing. The 5’-primers and 3’- antisense primer were unique for each isoforms. Variability measurement in vertical dimension showed stronger genetic component than sagittal. This study aims to obtain the genotype marker of vertical mandibular asymmetry related to PITX2A and PITX2D isoform by visualization of the amplified product on stained gel to allele specific oligonucleotide between the case and control with Restriction Fragment LengthPolymorphism (RFLP). Determination of vertical mandibular asymmetry based on condylar height asymmetry index of pre-treatment panoramic radiograph using Kjellberg’s technique whilst vertical mandibular growth pattern using lateral cephalogram. The differences of condylar height asymmetry in case-control based on vertical growth pattern was compared using Pearson’s chi-squared test. DNA extraction of 129 out-coming orthodontic patients in Universitas Sumatera Utara Dental Hospital were obtained from Buccal swab. Then DNA samples were amplified by Polymerase chain reaction (PCR) and digested with NciI restriction enzyme prior to electrophoresis visualization. There was no significant statistical difference in vertical mandibular asymmetry compared to vertical mandibular growth pattern. The RFLP analysis did not show any polymorphism for PITX2A and PITX2D isoform. All of the samples showed wild type homozygote. Further analysis method, except RFLP, were required to understand the genetic factor in the variance of vertical mandibular

Full Text Available Insulin-like growth factor binding protein-3 (IGFBP-3 gene is important for regulation of growth and development in mammals. The present investigation was carried out to study DNA polymorphism by PCR-RFLP of IGFBP-3 gene and its effect on fibre traits of Chinese Inner Mongolian cashmere goats. The fibre traits data investigated were cashmere fibre diameter, combed cashmere weight, cashmere fibre length and guard hair length. Four hundred and forty-four animals were used to detect polymorphisms in the hircine IGFBP-3 gene. A 316-bp fragment of the IGFBP-3 gene in exon 2 was amplified and digested with HaeIII restriction enzyme. Three patterns of restriction fragments were observed in the populations. The frequency of AA, AB and BB genotypes was 0.58, 0.33 and 0.09 respectively. The allelic frequency of the A and B allele was 0.75 and 0.25 respectively. Nucleotide sequencing revealed a C>G transition in the exon 2 region of the IGFBP-3 gene resulting in R158G change which caused the polymorphism. Least squares analysis revealed a significant effect of genotypes on cashmere weight (p0.05. The animals of AB and BB genotypes showed higher cashmere weight, cashmere fibre length and hair length than the animals possessing AA genotype. These results suggested that polymorphisms in the hircine IGFBP-3 gene might be a potential molecular marker for cashmere weight in cashmere goats.

Differentiation and classification of phytoplasmas have been primarily based on the highly conserved 16Sr RNA gene. RFLP analysis of 16Sr RNA gene sequences has identified 31 16Sr RNA (16Sr) groups and more than 100 16Sr subgroups. Classification of phytoplasma strains can however, become more refin...

To assess whether the biallelic -231 G>A polymorphism of the endothelin type A receptor (EDNRA) gene, previously shown to be a marker of increased risk for developing migraine, has a role in the susceptibility to primary pediatric headache. Several studies suggest that endothelin has a role in migraine. A recent association study has shown that the biallelic -231 G>A polymorphism of the EDNRA gene is associated to migraine in an elderly population. A total of 126 consecutive unrelated pediatric patients affected by primary headache, classified according to the International Headache Society criteria in migraine (migraine with aura, n = 3; migraine without aura, n = 80), and tension-type headache (episodic tension-type headache, n = 36; chronic tension-type headache, n = 7) patients, were recruited to the study. Sixty-seven healthy blood donors were used as a control group. Genomic DNA was extracted from buccal swabs or blood samples and analyzed by polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP) for the above-mentioned polymorphism. Allele and genotype frequencies for primary headache patients were analyzed in comparison with the control group. No significant differences were found in the distribution of the EDNRA -231 G>A polymorphic variant when considering both genotype (migraine chi2 = 2.78, P = .25; tension-type headache chi2 = 3.58, P = .17) and allelic frequencies (migraine chi2 = 1.48, P = .22; tension-type headache chi2 = 0.39, P = .56). Furthermore, no significant genotype-related difference was found in relation to clinical features, such as age at onset, frequency, and length of the attacks. Our study shows that the -231 G>A polymorphism in the EDNRA gene is neither associated with primary juvenile headache nor significantly correlated with main clinical features characteristic of the headache pathology in pediatric settings.

Full Text Available A recent genome-wide association study of lung cancer among never-smoking females in Asia demonstrated that the rs2736100 polymorphism in the TERT-CLPTM1L locus on chromosome 5p15.33 was strongly and significantly associated with risk of adenocarcinoma of the lung. The telomerase gene TERT is a reverse transcriptase that is critical for telomere replication and stabilization by controlling telomere length. We previously found that longer telomere length measured in peripheral white blood cell DNA was associated with increased risk of lung cancer in a prospective cohort study of smoking males in Finland. To follow up on this finding, we carried out a nested case-control study of 215 female lung cancer cases and 215 female controls, 94% of whom were never-smokers, in the prospective Shanghai Women's Health Study cohort. There was a dose-response relationship between tertiles of telomere length and risk of lung cancer (odds ratio (OR, 95% confidence interval [CI]: 1.0, 1.4 [0.8-2.5], and 2.2 [1.2-4.0], respectively; P trend = 0.003. Further, the association was unchanged by the length of time from blood collection to case diagnosis. In addition, the rs2736100 G allele, which we previously have shown to be associated with risk of lung cancer in this cohort, was significantly associated with longer telomere length in these same study subjects (P trend = 0.030. Our findings suggest that individuals with longer telomere length in peripheral white blood cells may have an increased risk of lung cancer, but require replication in additional prospective cohorts and populations.

Methylenetetrahydrofolate reductase (MTHFR) gene plays a pivotal role in folate metabolism. Several genetic variations in MTHFR gene as MTHFR-C677T and MTHFR-A1298C result in decreased MTHFR activity, which could influence efficient DNA methylation and explain susceptibility to different cancers. The etiology of chronic myeloid leukemia (CML) is obscure and little is known about individual's susceptibility to CML. In order to assess the influence of these genetic polymorphisms on the susceptibility to CML and its effect on the course of the disease among Egyptians, we performed an age-gender-ethnic matched case-control study. The study included 97 CML patients and 130 healthy controls. Genotyping of MTHFR-C677T and -A1298C was performed by polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP) technique. The results showed no statistical difference in the distribution of MTHFR-C677T and -A1298C polymorphic genotypes between CML patients and controls. The frequency of MTHFR 677-TT homozygous variant was significantly higher in patients with accelerated/blastic transformation phase when compared to those in the chronic phase of the disease. In conclusion, our study revealed that MTHFR-C677T and -A1298C polymorphisms could not be considered as genetic risk factors for CML in Egyptians. However, MTHFR 677-TT homozygous variant might be considered as a molecular predictor for disease progression.

Increased concentrations of tumor necrosis factor alpha (TNF-α) in blood and amniotic fluid are observed in women with preterm delivery (PTD) and TNF-α mutations at -308 position are associated with higher expression of this gene. Therefore, we compared the frequency of G308A transition in the promoter region of TNF-α gene of women and neonates delivered preterm with the normal subjects. This cross-sectional study was performed on 135 mothers who were referred for delivery. According to the gestational age, mothers and their neonates were allocated to the case (preterm, 64 subjects) and control (term, 71 subjects) groups. Using the polymerase chain reaction, restrictive fragment lengthpolymorphism (RFLP), genotyping was performed on both maternal peripheral blood and cord blood samples to determine single nucleotide polymorphism in the promoter region of TNF-α gene at -308. Two mothers in the case group, one mother in the control group and one neonate in the case group had genotyping assays (GA) mutation. All other subjects had normal GG genotype. Frequency of GA mutation was not significantly different between two groups (P = 0.47). There is no significant association between PTD and either maternal or fetal TNF-α -308 polymorphism and frequency ofGAmutation is not significantly increased in mothers and neonates delivered preterm. It means that the presence of this mutation by itself does not modify the overall risk of PTD. Investigations on the combination of various polymorphisms indifferent genes are recommended to achieve more accurate results.

Full Text Available Background and Objectives: Apolipoprotein A5 (APOA5 gene is important in determining plasma triglyceride levels, a major cardiovascular disease risk factor. Mutation in this gene affected plasma triglyceride level. We looked for possible associations of the APOA5 gene polymorphism S19W with coronary heart disease (CHD in a sample of Iranian population. Materials and Methods: A total of 73 CHD patients and 55 controls were genotyped by polymerase chain reaction–restriction fragment lengthpolymorphism (PCR-RFLP for this single nucleotide polymorphism. Serum lipids and Fast Blood Sugar concentrations were measured in all subjects with enzymatic method. Results: Allele frequencies observed in our population were 0.041 for the W allele and 0.959 for the S allele which are similar to other populations (p>0.05. There is no evidence that APOA5 S19W, is a risk factor of CHD in our sample (p>0.05. In addition, we observed no association between the APOA5 W allele and elevated plasma TG levels (p>0.05 in the CHD group. This result was also present in the control group (p>0.05. Conclusion: The APO A5 gene polymorphism in S19W gene has no association with the high susceptibility to CHD.

Full Text Available Abstract Background Tribbles 3 (TRB3 affects insulin signalling by inhibiting insulin-stimulated Akt phosphorylation and subsequent activation. A single nucleotide polymorphism located in the second extron of the human TRB3 gene is thought to be associated with insulin resistance. The latter is a core abnormality in PCOS independent of obesity. The present study was designed to clarify the relationships of TRB3 Q84R polymorphism with PCOS in a Chinese women group. Methods A case-control study with two groups: PCOS group (n = 336 and control group of infertility women for tubal and/or male factor (n = 116 was performed. Genotyping of the TRB3 R84 variant was determined by polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP. Results The frequency of genotype QQ in PCOS women was significantly lower, while genotype QR and RR were significantly higher than that in control group (p Conclusions TRB3 Q84R polymorphism is associated with obesity and especially glucose metabolism and not associated with polycystic ovary syndrome because of compositional characteristics of phenotype in Chinese PCOS women.

Full Text Available Biochemical polymorphism study is a method of determination of genetic variation. This variability could be a basis for selection and subsequent genetic improvement in farm animals. The polymorphism in the intron 1 of chicken growth hormone (cGH gene was investigated in the Iranian native fowls by using polymerase chain reaction (PCR-restriction fragment lengthpolymorphism (RFLP method. The genomic DNA was extracted from 217 samples (129 samples from the native fowls of Isfahan province and 88 samples from the native fowls of Mazandaran province by using modified salting out technique. The DNA fragment of the growth hormone gene with 776 bp was amplified by PCR using specific primers. Then the PCR products were digested with MspI restriction enzyme and analyzed on 2.5% agarose gel. The allelic frequency of intron 1 locus for A1, A2 and A3 alleles in Isfahan native fowls were 0.60, 0.21 and 0.19 and those in Mazandaran native fowls were 0.28, 0.05 and 0.67, respectively. The results of current study indicated that the intron 1 of cGH is polymorphic in Iranian native fowls and could be exploited as a candidate gene for marker-assisted selection for growth-related traits.

Full Text Available Growth hormone (GH is an anabolic hormone which sintesized and secreted by somatrotop cell inpituitary anterior lobe. GH exert its effect on growth and metabolism by interacting with a specificreceptor on the surface of the target cells. Growth hormone receptor (GHR has been suggested ascandidate gene for traits related to meat production in Bovidae. The objectives of this study were toidentify polymorphism of GH and GHR genes in buffalo. The 452 DNA samples buffalo were collectedfrom five populations in Indonesia (Siborong-Borong-Medan (65, Lebak-Banten (29, Pandeglang-Banten (180, Semarang-Central Java, and Mataram-West Nusa Tenggara (103. A gene fragment of theGH|AluI gene at 432 bp located on exon 3 and GHR|AluI gene at 298 bp on exon 10 were successfullyamplified by using the techniques of a PCR (polymerase chain reaction and genotyped by PCR-RFLP(restriction fragment lengthpolymorphism then -SSCP (single strand conformation polymorphism. Theresults showed no polymorphisms were detected in these genes. All buffaloes tested had LL genotype forlocus GH|AluI and AA genotype for locus GHR|AluI.

The organic solvents and other exogenous compounds of metabolic enzymes genetic variation may affect the risk of the toxic effect of organic solvents exposure. Therefore, this research we observed the glutathione transferase M1 and T1 (GSTM1, GSTT1) deletion mutation genotype, two kinds of microsomal epoxide hydrolase (mEPHX) genetic polymorphism, organic solvents exposure and smoking effection in chronic cases of toxic encephalopathy (CTE) correlation. The object was 115 patients who had a long history of organic solvents exposure, were divieded into two groups: CTE (n=83) , no CET (n=32) according to clinical diagnosis. DNA was isolated from patients in white blood cells through the multiple-polymerase chain reaction to determine the loss of GSTM1 and GSTT1 genotype. two kinds of mEPHX polymorphism were analysised through the PCR-RFLP (restriction fragment lengthpolymorphism). The relative risk has obviously improved when lack of GSTM1 genotypes to CTE (RR=2.35, 95% CI 2.35 0.96). in according to the patient's Smoking condition and classify genotype, patients lack of GSTM1 genotypes had a significantly higher risk CTE than GSTM1+genotype patients (RR=3.13, 95% CI 3.13 1.2) , both mEPHX polymorphisms had nothing to do with an increased risk of CTE. The GSTM1 genotypes played an important role in the organic solvent induced the CTE of susceptibility.it was Influenced by the interaction between smoking at the same time.

The clinical outcome to Leishmania-infection is determined by the individual adaptive immune T helper cell responses and their interactions with parasitized host cells. An early development of a proinflammatory immune response (Th1 response) is necessary for Leishmania-infection resolution. The Toll-interacting protein (TOLLIP) regulates human Toll-like receptors signaling pathways by down regulating the proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) and inducing the ant-inflammatory cytokine interleukin-10 (IL-10). Polymorphisms in the TOLLIP gene are associated with infectious diseases. The polymorphisms rs5743899 and rs3750920 in the TOLLIP gene were genotyped by polymerase chain reaction and restriction fragment lengthpolymorphism (RFLP) analysis in 631 patients with cutaneous leishmaniasis (CL) caused by L. guyanensis and 530 individuals with no history of leishmaniasis. The G and T alleles of the rs5743899 and rs3750920 were more common in patients with CL than in healthy individuals (P = 2.6 x10(-8) ; odds ratio [OR], 1.7 [ 95% confidence interval (CI) 1.4-2.0] and P = 1.9 x10(-8) ; OR, 1.6 [95% CI 1.4-1.9] respectively). The r2 and D' linkage disequilibrium between the two polymorphisms are 0.05 and 0.473 with a confidence bounds of 0.37 to 0.57 respectively. The two polymorphisms are independently associated with an increased risk of developing CL.

Full Text Available Abstract Background Rhinitis and asthma are very common diseases involving genetic and environmental factors. Most patients with asthma also have rhinitis, which suggests the concept of 'one airway, one disease.' A disintegrin and metalloproteinase 33 (ADAM33 is the first asthma-susceptible gene to be discovered by positional cloning. To evaluate the potential influence of ADAM33 gene polymorphisms on allergic rhinitis (AR and allergic asthma (AS, a case-control study was conducted on the Han population of northeast China. Methods Six polymorphic sites (V4, T+1, T2, T1, S1, and Q-1 were genotyped in 128 patients with AR, 181 patients with AS, and 151 healthy controls (CTR. Genotypes were determined by the polymerase chain restriction fragment lengthpolymorphism (PCR-RFLP method. Data were analyzed using the chi-square test with Haploview software. Results The single nucleotide polymorphisms (SNPs, V4 G/C, T+1 A/G, and T1 G/A, of the ADAM33 gene may be the causal variants in AR, whereas ADAM33 V4 G/C, T2 A/G, T1 G/A, and Q-1A/G may participate in the susceptibility of AS. Conclusion These results suggest that polymorphisms of the ADAM33 gene may modify individual susceptibility to AR and AS in a Chinese Han population.

Clinical studies have reported a significant association between matrix metalloproteinases (MMP), particularly (MMP-9), and inflammatory diseases including Behçet's disease (BD). To study the relationship between MMP-9 rs17576 gene polymorphism and the development of BD, and its relation to disease activity among Egyptian patients. A total of 100 BD patients and 100 healthy control volunteers were genotyped for MMP-9 rs17576 polymorphism with polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP), followed by the confirmation of our results in random subgroups using direct DNA sequencing technique. The frequency of the GG genotype and G allele was significantly higher in BD patients as compared to the normal controls (p = 0.011, OR 8.61; p = 0.03, OR 1.65, respectively). There was no significant association between the MMP-9 rs17576 polymorphism and the clinical outcomes of BD. our study suggests a significant association of the MMP-9 rs17576 A/G polymorphism with increased risk of BD development in Egyptian patients.

Full Text Available The promoter polymorphism −174G/C within the interleukin-6 gene (IL-6 has been reported to have a functional importance through the modulation of IL-6 gene expression in vitro and in vivo. IL-6 is thought to play an important role in autoimmune diseases and the effect of its receptor inhibitor—tocilizumab—has been recently studied. The aim of this case-control study was to investigate the association between the interleukin-6 −174G/C single nucleotide polymorphism and the susceptibility to dermatomyositis (DM and systemic lupus erythematosus (SLE in Bulgarian patients. Altogether, 87 patients—52 with SLE and 35 with DM—as well as 80 unrelated healthy controls were included in this study. All of them were analyzed by restriction fragment lengthpolymorphism analysis (RFLP. The GG genotype and the G allele appeared to be associated with SLE, especially in women. None of the genotypes showed an association with DM. However, the G allele appeared to be associated with muscle weakness and it is a risk factor for elevated muscle enzymes. Our results indicate that IL-6 −174G/C polymorphism might be associated with the susceptibility to SLE especially in women. Although it is not associated with DM, it seems that IL-6 −174G/C polymorphism could modulate some clinical features in the autoimmune myopathies.

Ovarian cancer is the leading cause of death from gynecological malignancies. The early stages of this disease are asymptomatic and more than 75% of the cases are diagnosed with regional or distant metastases. p53 gene is frequently mutated in some histological subtypes of ovarian carcinomas. The role of p53 mutations and polymorphic variant of codon 72 in the prognosis of disease is still unclear. The aim of this study was to determine the frequency of p53 mutations and polymorphic variants of codon 72 among ovarian carcinoma patients and to correlate them with clinicopathological characteristics of disease. 54 ovarian carcinoma patients were included in the study. DNA was isolated from tumor tissue by the salting- out method. p53 mutations in exons 4-8 were detected by PCR-SSCP (polymerase chain reaction - single-stranded conformational polymorphism) electrophoresis. Codon 72 polymorphism was assessed by RFLP (restriction fragment-lengthpolymorphism) method. p53 mutations were present in 11 out of 54 patients (20.4%). Twenty-four patients (44.4%) exhibited Arg/ Arg, 24 patients (44.4%) Arg/Pro and 6 patients (11.2%) Pro/ Pro genotype of 72 codon polymorphism. Correlations between p53 mutations and various clinicopathological characteristics were not found. However, we observed that the frequency of Pro/Pro genotype was increasing with higher histological grade as well as in advanced compared to localized disease, but without statistical significance. Distribution of p53 gene mutations between Pro/Pro genotype and Arg/Pro plus Arg/Arg genotypes was not statistically significant. Our study suggests that Pro/Pro genotype of 72 codon polymorphism could be an independent prognostic marker in ovarian carcinomas.

Herein, we investigated potential associations between polymorphisms of genes related to estrogen metabolism and bone mineral density (BMD) in postmenopausal women. This was a cross-sectional study, in which two hundred and ninety postmenopausal Mexican-Mestizo women were studied. The BMD of the lumbar spine (LS), total hip (TH), and femoral neck (FN) was measured. The distribution of the genetic polymorphisms, including rs1799814 and rs1048943 at CYP1A1 as well as rs1056836 at CYP1B1, were analyzed by polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP), single-stranded conformational polymorphism (SSCP), and DNA sequencing. Deviations from Hardy-Weinberg equilibrium (HWE) were tested, and linkage disequilibrium (LD) was calculated by direct correlation (r 2 ). Moreover, haplotype analysis was performed. All polymorphisms were in HWE. The genotype and allele distributions of the three single nucleotide polymorphisms (SNPs) studied showed no significant differences. However, statistical significance was reached when constructing haplotypes. The CG haplotype in CYP1A1 was associated with variations in LS and FN BMD after adjustment for covariates (p = 0.021 and 0.045, respectively), but the association with TH BMD was not significant. These results suggested that the CG haplotype in CYP1A1 may play an important role in the mechanism of osteoporosis and may be useful as a genetic marker.

Type 2 diabetes mellitus (T2DM) is growing in an epidemic manner across the world and India has the world's largest number of diabetic subjects. The present study was carried out to investigate the association of glutathione-S-transferase (GSTM1, GSTT1) and fat mass and obesity associated (FTO) gene polymorphisms with T2DM patients and controls, and its role in increasing the susceptibility to T2DM. A total of 198 subjects (101 T2DM patients and 97 controls) participated in this study. GSTM1, GSTT1 and FTO gene polymorphisms in the patients and controls were evaluated by polymerase chain reaction (PCR) and restriction fragment lengthpolymorphism (RFLP). We observed significant association of GSTM1 positive (p = 0.046) and GSTM1 null (p = 0.046) genotypes with T2DM, while no significant association was found with the FTO gene polymorphism in our study. It seems that the GSTM1 gene polymorphism can be a predictive marker for early identification of a population at risk of T2DM. The potential role of GST and FTO gene polymorphisms as a marker of susceptibility to T2DM needs further studies in a larger number of patients.

Full Text Available Type 2 diabetes mellitus (T2DM is growing in an epidemic manner across the world and India has the world’s largest number of diabetic subjects. The present study was carried out to investigate the association of glutathione-S-transferase (GSTM1, GSTT1 and fat mass and obesity associated (FTO gene polymorphisms with T2DM patients and controls, and its role in increasing the susceptibility to T2DM. A total of 198 subjects (101 T2DM patients and 97 controls participated in this study. GSTM1, GSTT1 and FTO gene polymorphisms in the patients and controls were evaluated by polymerase chain reaction (PCR and restriction fragment lengthpolymorphism (RFLP. We observed significant association of GSTM1 positive (p = 0.046 and GSTM1 null (p = 0.046 genotypes with T2DM, while no significant association was found with the FTO gene polymorphism in our study. It seems that the GSTM1 gene polymorphism can be a predictive marker for early identification of a population at risk of T2DM. The potential role of GST and FTO gene polymorphisms as a marker of susceptibility to T2DM needs further studies in a larger number of patients.

Full Text Available Youchao Jia,1,2 Aimin Zang,2 Shunchang Jiao,1 Sumei Chen,1 Fu Yan1 1Department of Medical Oncology, General Hospital of Chinese PLA, Beijing, 2Department of Oncology, Affiliated Hospital of Hebei University, Hebei, People’s Republic of China Abstract: The purpose of the present study was to determine the relationship between interleukin-18 (IL-18 -607 A/C polymorphism and the risk of non-small-cell lung cancer (NSCLC and its impact on the serum IL-18 level. The genotyping of IL-18 -607 A/C polymorphism was detected by polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP. The results showed that the AA/AC genotype distribution in NSCLC patients was significantly higher than that of healthy controls (P=0.02. However, no significant differences were found between the two subgroups when stratified by clinical characteristics. Furthermore, serum IL-18 levels were found to be significantly higher in the NSCLC patients than in the controls (P=0.01 as detected by enzyme-linked immunosorbent assay analysis. There was no correlation between serum IL-18 levels and different genotypes. In conclusion, these findings suggest that IL-18 -607 A/C polymorphism increases the risk of NSCLC in the Chinese population, and this polymorphism could not functionally affect the IL-18 levels. Keywords: IL-18, polymorphism, NSCLC

The vascular endothelial growth factor (VEGF), a major angiogenic factor, is known to play an important role in the development of endometriosis. The aim of this study was to investigate the association of three VEGF (-460 C/T, +405 G/C, and +936 C/T) polymorphisms with the risk of endometriosis in the Tunisian population. This study includes 105 women with endometriosis and 150 women with no laparoscopic evidence of disease. Genotyping of the VEGF -460 C/T, +405 G/C, and +936 C/T polymorphisms were performed by polymerase chain reaction restriction fragment lengthpolymorphism (PCR-RFLP). The distribution of genotypes (P = 0.006) and allele (P = 0.0009) frequencies of the +936 C/T polymorphism was significantly different between patients and controls. Patients with stages III-IV endometriosis showed a higher VEGF + 936T allele frequency than controls (P = 0.0001). However, the distribution of genotypes and allele frequencies of the VEGF -460 C/T and +405 G/C polymorphisms did not differ significantly between endometriosis patients and controls. These findings suggest that the VEGF +936 C/T polymorphism may be a risk factor for endometriosis development and the VEGF +936 T allele is associated with an increased risk of stages III-IV endometriosis in the Tunisian population.

Several reports on science education suggest that students at all levels learn better if they are immersed in a project that is long term, yielding results that require analysis and interpretation. I describe a 12-wk laboratory project suitable for upper-level undergraduates and first-year graduate students, in which the students molecularly locate and map a gene from Drosophila melanogaster called dusky and one of dusky's mutant alleles. The mapping strategy uses restriction fragment length ...

Glutathione S-transferases (GSTM1, GSTT1, and GSTP1) and methylenetetrahydrofolate reductase (MTHFR) are important enzymes for protection against oxidative stress. In addition, MTHFR has an essential role in DNA synthesis, repair, and methylation. Their polymorphisms have been implicated in the pathogenesis of ulcerative colitis (UC). The aim of the present study was to investigate the role of selected polymorphisms in these genes in the development of UC in the Moldavian population. In a case-control study including 128 UC patients and 136 healthy individuals, GSTM1 and GSTT1 genotypes (polymorphic deletions) were determined using multiplex polymerase chain reaction (PCR). The GSTP1 rs1695 (Ile105Val), MTHFR rs1801133 (C677T), and MTHFR rs1801131 (A1298C) polymorphisms were studied with restriction fragment lengthpolymorphism (RFLP) analysis. Genotype-phenotype correlations were examined using logistic regression analysis. None of the genotypes, either alone or in combination, showed a strong association with UC. The case-only sub-phenotypic association analysis showed an association of the MTHFR rs1801133 polymorphism with the extent of UC under co-dominant (p corrected = 0.040) and recessive (p corrected = 0.020; OR = 0.15; CI = 0.04-0.63) genetic models. Also, an association between the MTHFR rs1801131 polymorphism and the severity of UC was reported for the over-dominant model (p corrected = 0.023; coefficient = 0.32; 95% CI = 0.10-0.54). The GST and MTHFR genotypes do not seem to be a relevant risk factor for UC in our sample. There was, however, evidence that variants in MTHFR may influence the clinical features in UC patients. Additional larger studies investigating the relationship between GST and MTHFR polymorphisms and UC are required.

Full Text Available The objective of the present study was to estimate the allelic and genotypic frequencies of the polymorphisms E2FB (AY138588.1: c.305C> T, located in the leptin gene (LEP, and TG5 (X05380.1:g.-422C>T, located in the thyroglobulin gene (TG, and evaluate the association of these polymorphisms in crossbred cattle of seven distinct genetic groups with the following traits: slaughter weight (SW, hot carcass weight (HCW, hot carcass yield (HCY, carcass fat thickness (CFT, ribeye area (REA, marbling (MARM and shear force (SF. The animals were genotyped using the PCR-RFLP (Polymorphism Chain Reaction-Restriction Fragment LengthPolymorphism technique, using 201 products obtained from F1 Caracu × Nellore, Angus × Nellore and Valdostana × Nellore cows, mated to Canchim, Caracu and Red Angus bulls (only Caracu × Nellore cows were used with Red Angus bulls. The allelic and genotypic frequencies were compared using the Chi-squared test. Associations between the genotype of each polymorphism and the traits were analyzed using the General Linear Model (GLM of statistical software SAS. The least squares means of genotypes of the polymorphisms were compared using Student's t test. The E2FB polymorphism in the LEP gene was associated with CFT, showing the potential for use in national programs for genetic improvement of beef cattle, through the inclusion of SNP in genotyping commercial tests. The TG5 polymorphism in the TG gene was not associated with any of the evaluated traits and was considered ineffective for selection of beef cattle in Brazilian herds.

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Association of polymorphisms in avian apoVLDL-II gene with body weight and abdominal fat weight. HH Musa, GH Chen ... Blood samples from the respective populations were taken for DNA extraction, and then slaughter for fat determination. Polymorphism was detected by PCR-RFLP and PCR-SSCP techniques.

Full Text Available Background: Single nucleotide polymorphism (SNPs are considered as one of the underlyingcauses of male infertility. Proper sperm chromatin packaging which involves replacement ofhistones with protamines has profound effect on male fertility. Over 20 SNPs have been reportedfor the protamine 1 and 2.Materials and Methods: The aim of this study was to evaluate the frequency of two previouslyreported SNPs using polymerase chain reaction (PCR-restriction fragment lengthpolymorphism(RFLP approach in 35, 96 and 177 normal, oligozoospermic and azoospermic individuals. TheseSNPs are: 1. A base pair substitution (G at position 197 instead of T in protamine type 1 Openreading frame (ORF including untranslated region, which causes an Arg residue change to Serresidue in a highly conserved region. 2. cytidine nucleotide change to thymidine in position of 248of protamine type 2 ORF which caused a nonsense point mutation.Results: The two mentioned SNPs were not present in the studied population, thus concluding thatthese SNPs can not serves as molecular markers for male infertility diagnosis.Conclusion: The results of our study reveal that in a selected Iranian population, the SNP G197Tand C248T are completely absent and are not associated with male infertility and therefore theseSNPs may not represent a molecular marker for genetic diagnosis of male infertility.

Allozyme and mitochondrial DNA (mtDNA) restriction studies were undertaken to determine the extent of genetic divergence among field populations of Anopheles albitarsis in Brazil. Two sympatric species, An. deaneorum and An. marajoara, were identified in collections from Costa Marques (CM), Rondonia. Genetic evidence includes (1) the presence of two types of individuals, each with diagnostic allelic clusters (for Had-1, Pgi-1, Pep-1, Mpi-1, and Idh-1), (2) a deficiency of heterozygotes, and (3) characteristic mtDNA haplotypes. In addition, two allopatric cryptic species of An. marajoara were identified, one from Iguape (An. marajoara form IG), Sao Paulo state, and the other from the Island of Marajo (An. marajoara form MA). Though form IG and form-MA resemble form CM in wing spot morphology, they differ from it in diagnostic allozymes and mtDNA haplotypes. An. marajoara form CM had a higher variability (mean heterozygosity, H = 0.22, and percentage of polymorphic loci, P = 66.7) than did form IG and form MA (H = 0.08 in both, and P = 25.0 and 33.3, respectively). Form MA and form IG are genetically more similar to each other than both are to form CM. Based on wing morphology, estimates of F statistics, and genetic similarities, we propose that An. albitarsis in Brazil is a species complex. It comprises at least two morphologically distinguishable species: (1) An. deaneorum (currently one taxon) and (2) the An. marajoara species complex, which further consists of at least three cryptic forms, marajoara form MA, marajoara form IG, and marajoara form CM.

Objectives Genomic copy number variations (CNVs) are a major form of variation in the human genome and play an etiologic role in several neuropsychiatric diseases. Tandem repeats, particularly with long (> 50bp) repeat units, are a relatively common yet underexplored type of CNV that may significantly contribute to human genomic variation and disease risk. We therefore performed a pilot experiment to explore the potential role of long tandem repeats as risk factors in psychiatric disorders. Methods A bacterial artificial chromosome (BAC)-based array comparative genomic hybridization (aCGH) platform was used to examine CNVs in genomic DNA from 34 probands with schizophrenia or schizoaffective disorder. Results The aCGH screen detected an apparent deletion on 5p15.1 in two probands, caused by the presence in each proband of two low copy number (short) alleles of a tandem repeat that ranges in length from 50 3.4 kb units in the population examined. Short alleles partially segregate with schizophrenia in a small number of families, though linkage was not significant. An association study showed no significant difference in repeat length between 406 schizophrenia cases and 392 controls. Conclusion Though we did not demonstrate a relationship between the 5p15.1 repeat and schizophrenia, our results illustrate that long tandem repeats represent an intriguing type of genetic variation that have not been previously studied in connection with psychiatric illness. aCGH can detect a small subset of these repeats, but systematic investigation will require the development of specific arrays and improved analytic methods. PMID:19672138

Coronary artery disease (CAD) was the second leading cause of death during the last 3 years in Taiwan. Smooth muscle cells, monocytes/macrophages, and endothelial cells produce monocyte chemoattractant protein-1 (MCP-1) within atherosclerotic plaques following binding to the chemokine receptor-2 (CCR-2). Previous studies have well-documented the association between MCP-1 expression and susceptibility to, or clinicopathological features, of CAD. This study investigated the relationships between MCP-1-2518A/G and CCR-2-V64I genetic polymorphisms and CAD in the Taiwanese population. A total of 608 subjects, including 392 non-CAD controls and 216 patients with CAD, were recruited and subjected to polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP) to evaluate the effects of these two polymorphic variants on CAD. Results indicated a significant association between MCP-1 -2548 gene polymorphism and susceptibility to CAD. GG genotypes (OR = 1.629; 95 % CI = 1.003-2.644), or individuals with at least one G allele (OR = 1.511; 95 % CI = 1.006-2.270), had a higher risk of CAD as compared with AA genotypes. Results also revealed that subjects with at least one A allele of the V64I CCR2 gene polymorphism had significantly increased risk of CAD. G allele in MCP-1-2518 might contribute to higher prevalence of atrial fibrillation in CAD patients (OR = 4.254; p CCR-2 64I gene polymorphisms represent important factors in determining susceptibility to CAD, and the contribution of MCP-1-2518G could be through effects on atrial fibrillation in CAD patients.

Full Text Available We sampled 119 Nelore cattle (Bos indicus, 69 harboring B. indicus mtDNA plus 50 carrying Bos taurus mtDNA, to estimate the frequencies of putative mtDNA single nucleotide polymorphisms (SNPs and investigate their association with Nelore weight and scrotal circumference estimated breeding values (EBVs. The PCR restriction fragment lengthpolymorphism (PCR-RFLP method was used to detect polymorphisms in the mitochondrial asparagine, cysteine, glycine, leucine and proline transporter RNA (tRNA genes (tRNAasn, tRNAcys, tRNAgly, tRNAleu and tRNApro. The 50 cattle carrying B. taurus mtDNA were monomorphic for all the tRNA gene SNPs analyzed, suggesting that they are specific to mtDNA from B. indicus cattle. No tRNAcys or tRNAgly polymorphisms were detected in any of the cattle but we did detect polymorphic SNPs in the tRNAasn, tRNAleu and tRNApro genes in the cattle harboring B. indicus mtDNA, with the same allele observed in the B. taurus sequence being present in the following percentage of cattle harboring B. indicus mtDNA: 72.46% for tRNAasn, 95.23% for tRNAleu and 90.62% for tRNApro. Analyses of variance using the tRNAasn SNP as the independent variable and EBVs as the dependent variable showed that the G -> T SNP was significantly associated (p < 0.05 with maternal EBVs for weight at 120 and 210 days (p < 0.05 and animal's EBVs for weight at 210, 365 and 455 days. There was no association of the tRNAasn SNP with the scrotal circumference EBVs. These results confirm that mtDNA can affect weight and that mtDNA polymorphisms can be a source of genetic variation for quantitative traits.

Full Text Available Background & objectives: The Vitamin-D receptor (VDR regulates vitamin D levels and calcium metabolism in the body and these are known to be associated with endocrine dysfunctions, insulin resistance and type-2 diabetes in polycystic ovarian syndrome (PCOS. Studies on VDR polymorphisms among PCOS women are sparse. We undertook this study to investigate the association pattern of VDR polymorphisms (Cdx2, Fok1, Apa1 and Taq1 with PCOS among Indian women. Methods: For the present study, 250 women with PCOS and 250 normal healthy control women were selected from Hyderabad city, Telangana, India. The four VDR polymorphisms were genotyped and analysed using ASM-PCR (allele specific multiple PCR and PCR-RFLP (restriction fragment lengthpolymorphism. Results: The genotype and allele frequency distributions of only Cdx2 showed significant difference between the PCOS cases and control women, indicating protective role of this SNP against PCOS phenotype. However, significant association was observed between VDR genotypes and some of the PCOS specific clinical/biochemical traits. For example, Fok1 showed a significant genotypic difference for the presence of infertility and Cdx2 genotpes showed association with testosterone levels. Further, the two haplotypes, ACCA and ACTA, were found to be significantly associated with PCOS indicating haplotype specific risk. Interpretation & conclusions: Although VDR polymorphisms have not shown significant association with PCOS, in view of functional significance of the SNPs considered, one cannot yet rule out the possibility of their association with PCOS. Further, specifically designed studies on large cohorts are required to conclusively establish the role of VDR polymorphisms in PCOS, particularly including data on vitamin D levels.

Full Text Available Nanociliates have been frequently found to be important players in the marine microbial loop, however, little is known about their diversity and distribution in coastal ecosystems. We investigated the molecular diversity and distribution patterns of nanoplanktonic oligotrich and choreotrich (OC ciliates in surface water of three neritic basins of northern China, the South Yellow Sea (SYS, North Yellow Sea (NYS, and Bohai Sea (BS in June and November 2011. SSU rRNA gene clone libraries generated from three summertime samples (sites B38, B4 and H8 were analyzed and revealed a large novel ribotype diversity, of which many were low-abundant phylotypes belonging to the subclass Oligotrichia, but divergent from described morphospecies. Based on the data of terminal-restriction fragment lengthpolymorphism (T-RFLP analysis of all 35 samples, we found that the T-RF richness was generally higher in the SYS than in the BS, and negatively correlated with the molar ratio of P to Si. Overall, multidimensional scaling and permutational multivariate analysis of variance of the community turnover demonstrated a distinct seasonal pattern but no basin-to-basin differentiation across all samples. Nevertheless, significant community differences among basins were recognized in the winter dataset. Mantel tests showed that the environmental factors, P:Si ratio, water temperature and concentration of dissolved oxygen (DO, determined the community across all samples. However, both biogeographic distance and environment shaped the community in winter, with DO being the most important physicochemical factor. Our results indicate that the stoichiometric ratio of P:Si is a key factor, through which the phytoplankton community may be shaped, resulting in a cascade effect on the diversity and community composition of OC nanociliates in the N-rich, Si-limited coastal surface waters, and that the Yellow Sea Warm Current drives the nanociliate community, and possibly the

Nanociliates have been frequently found to be important players in the marine microbial loop, however, little is known about their diversity and distribution in coastal ecosystems. We investigated the molecular diversity and distribution patterns of nanoplanktonic oligotrich and choreotrich (OC) ciliates in surface water of three neritic basins of northern China, the South Yellow Sea (SYS), North Yellow Sea (NYS), and Bohai Sea (BS) in June and November 2011. SSU rRNA gene clone libraries generated from three summertime samples (sites B38, B4 and H8) were analyzed and revealed a large novel ribotype diversity, of which many were low-abundant phylotypes belonging to the subclass Oligotrichia, but divergent from described morphospecies. Based on the data of terminal-restriction fragment lengthpolymorphism (T-RFLP) analysis of all 35 samples, we found that the T-RF richness was generally higher in the SYS than in the BS, and negatively correlated with the molar ratio of P to Si. Overall, multidimensional scaling and permutational multivariate analysis of variance of the community turnover demonstrated a distinct seasonal pattern but no basin-to-basin differentiation across all samples. Nevertheless, significant community differences among basins were recognized in the winter dataset. Mantel tests showed that the environmental factors, P:Si ratio, water temperature and concentration of dissolved oxygen (DO), determined the community across all samples. However, both biogeographic distance and environment shaped the community in winter, with DO being the most important physicochemical factor. Our results indicate that the stoichiometric ratio of P:Si is a key factor, through which the phytoplankton community may be shaped, resulting in a cascade effect on the diversity and community composition of OC nanociliates in the N-rich, Si-limited coastal surface waters, and that the Yellow Sea Warm Current drives the nanociliate community, and possibly the microbial food webs

Full Text Available Due to the epidemiological alteration in distribution of Candida species as well as significant increasing trend of either intrinsic or acquired in resistance of some of these fungi, the precise identification of Candida species is necessary for effective antifungal therapy and also for prevention of nosocomial infections. PCR-RFLP method is indicated to be a reliable, rapid and simple technique which is able to differentiate the Candida species. In the present study, we applied this method to evaluate the distribution of Candida species in patients affected with cutaneous candidiasis in the Guilan province. 896 clinical cutaneous samples were collected from different parts of skin and nail of suspected patients referred to clinical centers all over the Guilan province during 24 months. Samples were examined directly with 15% KOH and cultured on fungal specific media. Genomic DNA was extracted and the restriction enzyme Msp1 was applied for polymorphism analysis. Totally, 47 yeast strains were successfully isolated from different clinical samples and identified by conventional as well as PCR-RFLP methods. The results indicated that Candida albicans (36.17% was the most frequent species followed by C. parapsilosis (25.53%, C. tropicalis (19.14%, C. guilliermondii (14.89%, C. famata (2.12% and C. krusei (2.12%. Female finger nails were the most common location to be affected by Candida species. In conclusion, PCR-RFLP method was successfully used for recognition of clinical Candida species within the Guilan province and obtained results revealed C. albicans as the predominant causative agent of cutaneous candidiasis. However, distribution of other Candida species did not completely consist with the reported distribution of Candida species in other parts of Iran with different climate to the Guilan province.

Hepatocellular carcinoma (HCC) is the fourth most common cancer worldwide, the main etiological factors being chronic infections with hepatitis B and C viruses. Genetic polymorphic forms of glutathione-S-transferase (GST) and microsomal epoxide hydrolase (mEPHX) have been associated with risk for various malignancies. The present study was undertaken to evaluate the association of GSTT1 and GSTM1 null genotypes and mEPHX polymorphisms with hepatitis virus-related HCC risk in an Indian population. Three groups of subjects were considered, control (n = 169), chronic viral hepatitis (n = 174), and HCC (n = 63). Polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP) was used for this polymorphic study. Genotype distributions between categories were compared using the chi2 test; odds ratios (ORs) and 95% confidence interval were calculated to express the relative risk. GSTT1 null genotype was associated with 2.23-fold (p GST and mEPHX variants share a positive association with viral-related HCC risk in Indian population, although a larger sample size is still required to confirm the results.

Dopamine D2 receptor is involved in reward-mediating mesocorticolimbic pathways. It plays an important role in major depressive disorder (MDD). Three gene polymorphisms Taq1A, C957T and -141C ins/del, were identified in the DRD2 gene among the Western population. These variants in the DRD2 gene might be associated with the susceptibility of MDD patients through affecting the bioeffects of endogenous dopamine neurotransmission. However, little is known about their occurrence in Chinese population and their association with the susceptibility of patients with major depressive disorder. In this study, a total of 338 unrelated adult Chinese Han population, including 224 healthy volunteers and 114 patients with major depressive disorder, were recruited. DRD2 polymorphisms (Taq1A and -141C ins/del) were detected using restriction fragment lengthpolymorphism (RFLP) analysis and the C957T were detected by sequencing directly. As a result, three polymorphisms were identified in Chinese Han population and all were common SNP. However, we could detect no evidence of genetic association between 3 markers in DRD2 and major depressive disorder in the Chinese Han population. To conclude, this result suggests that Taq1A, C957T and -141C ins/del of DRD2 gene may not be associated with major depressive disorder, also may be the sample sizes too small to allow a meaningful test.

Methylenetetrahydrofolate reductase (MTHFR) plays a central role in converting folate to methyl donor for DNA methylation. Because MTHFR is a key enzyme in folate metabolism, changes in its activity resulting from polymorphisms in the MTHFR gene could modify the susceptibility to cancer. Recently, the C677T and A1298C mutations of MTHFR were discovered to be associated with susceptibility in acute lymphoblastic leukemia (ALL). The association between MTHFR polymorphisms and susceptibility and clinical outcome in ALL was studied in 118 adult ALL patients and matched healthy controls (n =427). DNA samples taken from patients with ALL and controls were analyzed using polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP) assays to detect the MTHFR C677T and A1298C mutations. No significant difference was found in the development of adult ALL among those with different MTHFR genotypes of the C677T or A1298C polymorphisms. However, the MTHFR 677CT+TT genotype showed a tendency to be associated with adult ALL [crude odds ratio (OR), 0.67; 95% confidence interval (CI), 0.44-1.02; adjusted OR, 0.74 95% CI, 0.47-1.14]. The MTHFR C677T and A1298C polymorphisms are not significant risk factors in adult acute leukemia in the Korean population.